P2x receptor DNA and protein sequence

ABSTRACT

The P 2X  receptor of ATP has been cloned and expressed by recombinant DNA technology, so the receptor can be prepared free from other ATP receptors. The P 2X  receptor enables antibodies to be prepared and is useful in screening compounds for use in a variety of diseases and conditions, including epilepsy, cognition, emesis, pain (especially migraine), asthma, peripheral vascular disease, hypertension, diseases of the immune system, irritable bowel syndrome and premature ejaculation.

This invention relates to the P₂ X-purinoceptor, its preparation anduses.

The P₂ X-purinoceptor is a ligand-gated ion channel; that is, thereceptor itself forms an ion channel which opens when extracellularadenosine 5'-triphosphate (ATP) binds to the receptor. There are fiveother classes of neurotransmitter receptors (nicotinic acetylcholine,glutamate, glycine, GABA_(A) and 5-HT₃); these form a structurallyrelated superfamily of ligand-gated ion channels (Barnard, TrendsBiochem. Sci. 17, 368-374, (1992)). The P_(2x) -receptor now identifiesa new family of this type of receptor. The unique structure of thisreceptor, the widespread distribution of this receptor throughout thebody, and the numerous physiological roles this receptor may play, makeit an important protein that can be used to identify new,therapeutically effective, compounds for the treatment of a number ofpathological states.

In 1929 the eminent physiologist Szent-Gyorgyi described powerfulcardiovascular actions of extracellular purine nucleosides (e.g.adenosine) and nucleotides (e.g. ATP) (Drury & Szent-Gyorgyi, J.Physiol. 68 213-237 (1929)), but it was not until 1972 thatpharmacological evidence was provided to suggest the existence ofdistinct receptors for extracellular ATP (ie. that recognise ATP but notadenosine) (Burnstock, Pharmacological Reviews 21 509-581 (1972)). Theseminal and subsequent work on this area by Burnstock and colleagues waslargely unaccepted throughout the 1970s and early 1980s until thedevelopment of a range of relatively selective ligands and techniquesfor directly measuring ATP release overwhelmingly substantiatedBurnstock's hypothesis (Barnard et al., Trends Pharmacol. Sci. 15 67-70(1994)). In the past four or five years, unequivocal evidence for therole of ATP as a neurotransmitter has been provided for sympatheticcontrol of blood flow to the intestine and smooth muscle tone(contractility) in genitourinary tissue such as vas deferens, bladderand ureter (Barnard et al. (loc. cit.) and Evans & Surprenant, Brit. J.Pharmacol. 106 242-249 (1992)). Substantial indirect evidence alsoexists for the role of ATP as a neurotransmitter in a number of distinctneurones in the spinal cord, autonomic ganglia and certain nuclei in thecentral nervous system (Bean, Trends Pharmacol. Sci. 15 67-70 (1992),Evans et al., Nature 357, 503-505 (1992) and Edwards et al., Nature 359144-147 (1992)).

Purinoceptors are classified as P₁ (adenosine as ligand) and P₂ (ATP asligand). The P₂ receptors are subclassified into two broad types--thosethat are 7-transmembrane receptors that couple to G-proteins (P_(2Y),P_(2U), P_(2T), and perhaps P_(2Z)) and those that form a directly gatedion channel (P_(2X)). Pharmacological and/or physiological evidence forsubtypes of each of these types of receptors exists. The most recentnomenclature for these receptors is shown below.

    ______________________________________                                              P.sub.2X      P.sub.2Y    P.sub.2Z                                      ______________________________________                                        Type  Ligand-gated channel                                                                        G-protein coupled                                                                         Non-selective pore                              Subtype P.sub.2X1, P.sub.2X2, P.sub.2X3 P.sub.2Y1, P.sub.2Y2, P.sub.2Y3     ______________________________________                                    

Various P₂ receptors have previously been cloned. P_(2Y1) was cloned bythe Barnard/Burnstock group (Webb et al., FEBS Lett. 324 219-225 (1993))based on homology with other 7-TM G-protein coupled receptors. Thisgroup used PCR technology and primers based on conserved domains of thesecond and sixth transmembrane regions to screen a mammalian brain cDNAlibrary and, with final success, an embryonic chick whole-brain cDNAlibrary.

P_(2Y2) /P_(2U) was cloned by the Julius laboratory (Lustig et al.,Proc. Nat'l. Acad. Sci. USA 90 5113-5117 (1993)) by expression cloningin the oocyte from cDNA obtained from a NG108-15 neuroblastoma cellline.

P_(2Y3) /P_(2T) was also obtained by the Barnard/Burnstock group usingthe same probes and embryonic brain cDNA library used to obtain the P₂Y1 receptor (Barnard et al., Trends Pharmacol. Sci. 15 67-70 (1994)).

However, as yet, cloning of the P_(2X) receptor has remained an elusivegoal. The prior cloning exercises undertaken for the other P₂ receptorsdo not provide an adequate lead to enable the P_(2X) receptor to becloned. First, all the above purinoceptors are G-protein coupled 7-TMproteins. Their myriad functions (like those of all 7-TM receptors)occur through G-protein activation of one or more second messengersystems. There are over 200 currently identified proteins which belongto this 7-TM/G-protein coupled family. Agonists at these receptorsactivate cascades of intracellular transduction pathways, ofteninvolving several enzymes; the response of the cell is inherently slow(several seconds to minutes) and changes in excitability are subtle ifthey occur. In contrast, the P_(2X) receptor is a fundamentallydifferent type of purinoceptor that incorporates an ion channel.Activation of P_(2X) receptors is rapid (milliseconds), haspredominately local effects, and brings about immediate depolarisationand excitation.

Secondly, the tissue distribution of the P_(2X) receptor is distinctlydifferent from other purinoceptors, and the physiological roles differfrom other purinoceptors.

One of the principal established ways to clone a receptor is based onsequence relatedness of the nucleotides that encode the amino acids ofthe receptor protein; it depends on there being a fairly high level ofhomology between a known sequence and that of the unknown receptor. Thismethod was used to clone the P_(2Y1) form (above). Several laboratories,including that of the applicants, invested significant effort inobtaining the P_(2X) receptor using PCR techniques and primers based onconserved regions of various ligand-gated ion channels (ie. nicotinicACh, GABA, glutamate, 5-HT₃). This approach failed. With hindsight, thisfailure can be rationalised, as it can now, but only now, be seen thatthe structure of the P_(2X) receptor bears no homology with any of theseligand-gated ion channels. For the same reason, approaches based onfragment hybridisation would not succeed.

However, by adopting a different approach, it has now been foundpossible to clone the P_(2X) receptor, and it is on this achievementthat the present invention is in part based.

According to a principal aspect of the present invention, there isprovided a recombinant or isolated DNA molecule encoding a P_(2X)receptor, wherein the receptor:

(a) has the amino sequence shown in FIG. 1, FIG. 2, FIG. 3 or FIG. 4; or

(b) is substantially homologous to the sequence shown in FIG. 1, FIG. 2,FIG. 3 or FIG. 4;

or a fragment of such a DNA molecule, which fragment includes at least15 nucleotides taken from nucleotides 1 to 813 shown in FIG. 1, the fullnucleotide sequences shown in FIGS. 2 and 3, or from nucleotides 1 to1744 shown in FIG. 4.

The sequence shown in FIG. 1 is a cDNA sequence that encodes a rat vasdeferens P_(2X) receptor. This sequence is 1837 bases in length andencodes a protein of 399 amino acids. As was determined after thereceptor was cloned, approximately one half of the protein-encodingsequence, from nucleotides 814 onwards, had been discovered previouslybut the function of the previously cloned sequence was not known exceptthat it appeared to be implicated in apoptotic cell death (Owens et al.,Mol. Cell. Biol. 11 4177-4188 (1991)); the Owens et al sequence lacks atranslation initiation site and could not be made into protein. (In FIG.1, the upstream portion of the reported sequence of Owens et al., namelyPQLAHGCYPCPPHR, which is not shared with the P_(2X) receptor, is shownfor comparative purposes and does not form part of the invention.)

Preferably the FIG. 1 sequence fragments are taken from nucleotides1-810. Often the FIG. 4 sequence fragments are taken from nucleotides1-777.

The sequence shown in FIG. 2 is a cDNA sequence that encodes a ratsuperior cervical ganglion P_(2X) receptor.

The sequence shown in FIG. 3 is a cDNA sequence that encodes a ratdorsal root ganglion P_(2X) receptor.

The sequence shown in FIG. 4 is the cDNA sequence that encodes a humanP_(2X) receptor. The cDNA was isolated from the human urinary bladderusing a rat P_(2X) probe. It is 2643 bases long and encodes a 399 aminoacid protein having an amino acid sequence which is highly homologouswith the amino acid sequence of the rat P_(2X) receptor isolated fromrat vas deferens and with the rat P_(2X) receptors isolated from a ratsuperior cervical ganglion and from a rat dorsal root ganglion. Recentlywe have become aware of an expressed sequence tag corresponding toresidues 1745-1933 (Proc. Natl. Acad.Sci. USA 91,10645-10649 (Oct.1994).

Sequences which are substantially homologous to the FIG. 1, FIG. 2, FIG.3 or FIG. 4 amino acid sequence include those which encode proteinshaving at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% homology inincreasing order of preference. A protein having at least 99% homologywith the amino acid sequence of FIG. 1, FIG. 2, FIG. 3 or FIG. 4 willhave no more than four amino acid variations from such a sequence.Preferred substantially homologous sequences include P_(2X) sequencesfrom other species. Thus for the rat P_(2X) receptor sequences apreferred substantially homologous sequence is a human P_(2X) sequence.One method of determining sequence homology is disclosed in W R Pearsonand D J Lipman, Proc Natl Acad Sci USA 85:2444-2448 (1988).

Fragments may of course be larger than 15 nucleotides. Fragmentsencoding substantially the whole of the P_(2X) rat receptors or humanreceptor may be expected to share the biological activity of thereceptor, or at least some of its biological activities. Shorterfragments may be useful for encoding one or more selected domains of thereceptor, or simply as probes for detecting or identifying other usefulDNA sequences, including those encoding substantially homologousproteins. Fragments of at least 20, 30 or 50 nucleotides may be morefrequently of use than shorter ones.

DNA molecules of the invention are useful for a number of purposes.First, and not least, the P_(2X) cDNA shown in FIG. 1, in FIG. 2, inFIG. 3 and in FIG. 4 enables the relevant proteins to be expressed inliving cells. This would not be possible with fragments of the cDNA.However not only are fragments of DNA within the scope of the invention,for the various purposes mentioned above, but also genomic and othersequences of DNA (including synthetic DNA and "minigenes", which includeat least one, but not all, of the introns naturally present in the gene)are included within its scope. cDNA sequences encoding the rat receptorproteins or human P_(2X) receptor protein may be preferred in somecircumstances because such sequences are smaller than either genomic orminigene DNA and therefore more amenable to cloning manipulations. TheP_(2X) receptor protein can be stably expressible in chinese hamsterovary (CHO) cells, as will be described below.

Still on the subject of expression, while it would be possible toexpress genomic DNA in eukaryotic cells, it is much more difficult tomanipulate the DNA for insertion into host cells due to the larger sizethat commonly results from introns. The size is particularly importantfor the expression of RNA; very long cRNAs--the size of whole genes--aredifficult to make in sufficient quantity. On the other hand, expressionfrom RNA is much preferred at least for the investigation of ion channelproteins, because the Xenopus oocyte is sufficiently large to be studiedeasily by electro-physiological methods.

Secondly, the cDNA sequences encode proteins that, in their predictedfolding within the membrane, differ from other known proteins. This isadvantageous because, based on historical precedent, this will lead tothe discovery of a large family of related proteins and these may havefunctional roles unrelated to signalling mediated by ATP.

Thirdly, knowledge of the protein sequences encoded by rat and humanP_(2X) cDNA allows the development of molecular models that predict thedetailed disposition within the membrane. It further allows thecorrectness of such models to be determined by expression of mutagenisedproteins. These two approaches are advantageous because they may permitthe molecular design of complementary therapeutic agents that activateor block the receptor.

Fourthly, the P_(2X) cDNA sequences allow the distribution of the RNAthat encodes this receptor, as well as the receptor protein itself, tobe mapped in human tissues. RNA distribution can be determined by insitu hybridisation. Such hybridisation studies are disclosed in thepresent examples. Knowledge of a deduced amino acid sequence from cDNAallows synthetic peptides to be made that can be used to generateantibodies that selectively recognise a P_(2X) receptor. Thus a P_(2X)protein can be mapped by immunohistochemistry. This may suggest noveltherapeutic applications for drugs that activate or block the P_(2X)receptor, that can not be predicted on the basis of less sensitivecurrent methods for localising the receptor (radioactive ligandbinding).

Fifthly, rat P_(2X) cDNA is advantageous because it can allow theisolation of a closely related cDNA from human tissue.

Sixthly, the isolation of the human P_(2X) cDNA clone will enable ahuman genomic clone to be obtained. It is probable that mutations ofthis gene will be discovered that lead to human genetic disease. Theanalysis of such mutations may lead to appropriate treatments ofdiseases or disorders caused by such mutations.

In one aspect of the present invention rat vas deferens P_(2X) receptorwas cloned by a method which does not require prior inference aboutstructure. Tissues were chosen that were believed to be rich in the RNAfor the receptor of interest. A number of tissue sources were tried butthey did not provide RNA that led to ATP responses in oocytes.Eventually, vas deferens was chosen. From extracted polyadenylated RNA,a cDNA library or bank that corresponds as far as possible to the DNAsin the tissue was constructed. It was not assured, either before workbegan or until it was satisfactorily completed, that a satisfactory cDNAlibrary in which the rat P_(2X) gene was represented could beconstructed; nevertheless, this was achieved in plasmid pBKCMV.

An individual clone within the library that contains the rat vasdeferens P_(2X) cDNA of interest was detected by progressivefractionation of the library; at each step the fraction was tested todetermine whether RNA made from it can direct the formation of theprotein of interest. More specifically, RNA was transcribed in vitrofrom the cDNAs in the library (approximately 2 million) and the RNA("cRNA") mixture was injected into immature Xenopus oocytes. cRNA isvery susceptible to inadvertent enzymatic degradation, so all procedureswere carried out under sterile conditions. The cDNA pools were made bythe miniprep procedure and therefore contained large amounts of E. coliRNA; this difficulty was overcome by precipitating any RNA before thecRNA was transcribed.

Detection of the protein can in principle be done by radioactive ligandbinding or by a functional response. The activation of G proteins in theXenopus oocyte and the subsequent cellular response was used to obtainthe P_(2Y2) /P_(2U) receptor. In the present work, a decision was madeto use the opening of the integral ion channel of the P_(2X) as theresponse. Individual oocytes were screened two days after injection todetermine whether they had made P_(2X) receptor protein in theirmembrane. This was done by recording the current flowing across theoocyte membrane when ATP (30 μM) was applied to the outside of theoocyte; if the P_(2X) receptor has been produced, a small transientcurrent would be expected. However, testing for expression of thereceptor was not straightforward, as some batches of oocytes exhibitresponses to ATP because they naturally express other kinds of ATPreceptor. This difficulty was overcome as follows: when an oocyteresponded to ATP with the expected current this was further tested byblockade with a P_(2X) receptor antagonist (suramin). The cDNA fractionthat gave led to the positive response in such an oocyte was furtherdivided, and each fraction was again tested. Such progressivefractionation led to isolation of a single clone. The insert in theplasmid was sequenced; the sequence is shown in FIG. 1. This sequencewas used to design PCR primers which were used in the cloning of cDNAencoding a P_(2X) receptor from a rat superior cervical ganglion (seeFIG. 2). A similar procedure was then used in the cloning of cDNAencoding a P_(2X) receptor from a rat dorsal root ganglion (see FIG. 3).

DNA in accordance with the invention will usually be in recombinant orisolated form and may be in the form of a vector, such as a plasmid,phagemid, cosmid or virus, and in some embodiments contains elements todirect expression of the protein, for example in a heterologous host.Non-expressible vectors are useful as cloning vectors.

Although DNA in accordance with the invention may be preparedsynthetically, it is preferred that it be prepared by recombinant DNAtechnology. Ultimately, both techniques depend on the linkage ofsuccessive nucleotides and/or the ligation of oligo- and/orpoly-nucleotides.

The invention enables, for the first time, P_(2X) receptor to beprepared by recombinant DNA technology and hence free from protein withwhich it is naturally associated or contaminated (such as the P_(2U) or,particularly, P_(2Y) receptor, or other ATP receptors or bindingproteins), and this in itself forms another aspect of the invention. Theprotein will generally be associated with a lipid bilayer, such as acell, organelle or artificial membrane. P_(2X) receptor prepared byexpression of DNA in accordance with the first aspect may beglycosylated, but does not have to be. Generally speaking, receptorproteins and ion channels that are glycosylated will also function aftercarbohydrate removal or when expressed in cells that do not glycosylatethe protein. However, there are often important quantitative differencesin the function between the glycosylated and non-glycosylated protein.In the case of the rat vas deferens P_(2X) receptor, we believe that thenative protein is glycosylated because it has a molecular weight of 62kd when purified from the rat vas deferens, as compared to the molecularweight of 45 kd for the cloned protein. Similar results were obtainedfor the human P_(2X) receptor (see later).

There are also several asparagine residues in the extracellular domainthat are likely sites of sugar attachment.

Knowledge of the amino acid sequence of a P_(2X) receptor enables theprotein or peptide fragments of it to be prepared by chemical synthesis,if required. However, preparation by expression from DNA, or at leasttranslation from RNA, will usually be preferred.

Particularly useful peptide fragments within the scope of the inventioninclude epitopes (which may contain at least 5, 6, 7, 10, 15 or 20 aminoacid residues) of the P_(2X) receptor which are immunologicallynon-cross reactive with the RP-2 polypeptide disclosed in Owens et al.,loc. cit.

A P_(2X) receptor, and fragments of it, can be used to prepare specificpolyclonal and monoclonal antibodies, which themselves form part of theinvention. Polyclonal and monoclonal antibodies may be prepared bymethods well established in the art. Hybridoma and other cellsexpressing monoclonal antibodies are also within the invention.

RNA encoding a P_(2X) receptor, transcribable from DNA in accordancewith the invention and substantially free form other RNAs, also formspart of the invention, and may be useful for a number of purposesincluding hybridisation studies, in vitro translation and translation inappropriate in vivo systems such as Xenopus oocytes.

The invention also relates to host cells transformed or transfected witha vector as described above. Host cells may be prokaryotic or eukaryoticand include mammalian cells (such as COS, CHO cells and human embryonickidney cells (HEK 293 cells)), insect cells, yeasts (such asSaccharomyces cerevisiae) and bacteria (such as Escherichia coli). Hostcells may only give transient expression of the receptor, as in the caseof COS cells, but for preference the host cells are stably transfectedwith the vector. Host cells which appropriately glycosylate the receptorare preferred. A CHO cell line or any other cell line that stablyexpresses a P_(2X) receptor can be used for electrophysiological,calcium-influx, calcium-imaging and ligand-binding studies. Host cellswhich do not express the receptor may still be useful as cloning hosts.

A P_(2X) receptor prepared by recombinant DNA technology in accordancewith the invention has a number of uses, either in situ in a membrane ofthe expression host or in in vitro systems. In particular, the receptorcan be used as a screen for compounds useful in a variety of human (orother animal) diseases and conditions, as will now be briefly described.Such compounds include those present in combinatorial libraries, andextracts containing unknown compounds (e.g. plant extracts).

Epilepsy

Epilepsy results from overexcitation of distinct neurones in specificregions of the brain, in particular in the hippocampus. Functional ATPP_(2X) receptors are known to be present in some hippocampal neurones.If the P_(2X) receptors are expressed on inhibitory interneurons, thenreceptor agonists would be therapeutically useful. If the receptor isexpressed on principal (pyramidal or granule) cells, then receptorantagonists will be useful. If will now be possible to determine whichclasses of neuron express the receptor.

Cognition

Hippocampal neurones respond to ATP by activation of a P_(2X) receptor;these areas are of primary importance to cognition. It is now possibleto determine the cellular localisation of the P_(2X) receptor with inthe hippocampus; depending on this localisation, either agonists orantagonists might be effective to enhance memory.

Emesis

The acute trigger for emesis is rapid contraction of smooth muscle ofthe upper gastrointestinal tract. Activation of ATP P_(2X) receptorspresent on smooth muscle of the GI tract, in particular the stomach andtrachea, results in strong, rapid muscle contractions. P_(2X)-antagonists selective for visceral smooth muscle could be useful foremesis. Furthermore, P_(2X) receptors are known to be expressed in thenucleus of the tractus solitarius (Ueno et al., J. Neurophysiol. 68778-785 (1992)) and may be involved in transmission from primaryvisceral afferents; this could be blocked by selective P_(2X)antagonists.

Pain

First, P_(2X) receptors are expressed in dorsal horn neurones of thespinal cord. Activation of these neurones by ATP causes fastdepolarizing, excitatory responses (Jahr & Jessell, Nature 304 730-733(1983)); if a component of the transmission from nociceptive fibres ismediated by ATP then this could be blocked by a P_(2X) antagonist.Secondly, ATP is one of the most noxious substance known when appliedintradermally. This is because it activates directly the peripheralterminals of small diameter nociceptive fibres; it is known that thecell bodies in the dorsal root ganglion express P_(2X) receptors. AP_(2X) antagonist would be a peripherally active analgesic, and islikely to be effective in migraine.

Asthma

Bronchial smooth muscles contract in response to activation of P_(2X)receptors. This may occur in response to ATP released from sympatheticnerves, or from local immune cells. P_(2X) antagonists may help toprevent stimulus-evoked spasms of bronchial smooth muscle and therebyreduce the frequency and/or severity of asthmatic attacks.

Peripheral vascular disease

It is becoming clear that ATP and not noradrenaline is the primaryvasoconstrictor neurotransmitter in small resistance arteries--thosethat comprise over 70% of total peripheral resistance. This has beenshown for many vessels (Westfall et al., Ann. N.Y. Acad. Sci. 603300-310 (1991)). A selective antagonist could be used for localcollateral vaso-dilation.

Hypertension

Hypertension that is associated with increased sympathetic tone could betreated with P_(2X) receptor antagonists, because ATP is a majorexcitatory transmitter to many resistance vessels in several speciesincluding man (Westfall et al., loc. cit. and Martin et al., Br. J.Pharmacol. 102 645-650 (1991)).

Diseases of the immune system

A molecule identical to part of the P_(2X) receptor has been cloned fromthymocytes that have been induced to die (Owens et al., loc. cit.).

The selective expression in these conditions implies that a moleculeclosely related to the P_(2X) receptor plays a role in the apoptosisthat is an integral part of the selection of immunocompetent cells. Themolecule described by Owens et al. (RP-2) was incomplete and could nothave been translated into protein. The cloning of the P_(2X) receptorwill now allow the isolation of full length RP-2 clones, theirheterologous expression and the determination of their functional roles.

Irritable bowel syndrome

ATP is an important transmitter to the smooth muscles of the intestinaltract, particularly in the colon. It is also a transmitter betweenneurons in the enteric nervous system, by activating P_(2X) receptors(Galligan, Gastroenterology, in press). Antagonists at P_(2X) receptorsmay therefore have utility in the management of this condition.

Premature ejaculation

This could be prevented by preventing stimulus-evoked contraction of vasdeferens smooth muscle. P_(2X) receptors are highly expressed in thistissue; antagonists at this site would prevent vas deferenscontractility during sympathetic excitation.

Cystitis

P_(2X) receptors may be implicated in increased bladder sensitivity inpatients with cystitis. Thus antagonists of such P_(2X) receptors may beuseful in treating cystitis.

Useful agonists and antagonists identified as described above also forman aspect of the invention.

The cloning of the hP_(2X) receptor is an important aspect of thepresent invention. hP_(2X) is the first human member of a multigenefamily of ionotropic purinoceptors. Its strong similarity with P_(2X),isolated from rat vas deferens and with P_(2X) isolated from ratsuperior cervical ganglion or from rat dorsal root ganglion, suggeststhat it is a human homolog of the rat proteins. The present inventorshave found that differences between these two sequences are nearly allconservative substitutions of hydrophilic residues. Surprisingly,hP_(2X) has only 41% identity with the other reported P_(2X) receptor,that from rat PC12 cells (Brake et al, New structural motif forligand-gated ion channels defined by an ionotropic ATP receptor Nature371: 519-523 (1994)). The PC12 derived receptor was proposed to have asimilar membrane topography and shares the conserved spacing of cysteineresidues, indicated for the two smooth muscle sequences in FIG. 5.

The computed molecular weight of the hP_(2X) polypeptide (45 kd) agreeswith that of the in vitro translation product when made in absence ofpancreatic microsomal membranes. A larger product, 60 kd, produced inpresence of microsomes suggests glycosylation and supports the idea of acentral extracellular domain. The predicted hP_(2X) protein thus has thegeneral features of other cloned members of this family (Valera et al, Anew class of ligand-gated ion channel defined by P_(2X) receptor forextracellular ATP Nature 371: 516-519 (1994); Brake--supra): a large,cysteine-rich extracellular central domain flanked by two transmembranespans and short internal N- and C-termini.

The distribution of the hP_(2X) mRNA was examined by northern blotanalysis. Hybridisation of a principal 2.6 kb species was seen in allRNA samples tested, with the exception of brain. A smaller, 1.8 kb band,observed in spleen, and lung mRNAs could be due to a shorter 3'untranslated portion of the mRNA, as occurs for P_(2X) mRNA from the ratvas deferens. The hybridisation observed in thymus, lung, spleen andliver RNA may reflect the content of smooth muscle in those organs.However, hP_(2X) is likely to have roles in other cell types, asdemonstrated by its presence in adrenal gland, and the hemopoetic cellline HL60. The strong induction of hP_(2X) mRNA by HL60 differentiationmay reflect a parallel observation in rat in which the smooth muscleform of P_(2X) mRNA can be induced in immature thymocytes bydexamethasone (RP₂ mRNA; Owens et al, Identification of mRNAs associatedwith programmed cell death in immature thymocytes J J Molec Cell Biol11: 4177-4188 (1991)).

The present invention has enabled the first comprehensivepharmacological characterization of a cloned P_(2X) -purinoceptor to bemade. The time course of the responses to ATP and the sensitivity toα,β,-methylene ATP are similar to those reported for the native hP_(2X)in urinary bladder (Inoue & Brading, Human, pig and guinea-pig bladdersmooth muscle cells generate similar inward currents in response topurinoceptor activation Br J Pharmacol 103: 1840-1841 (1991)). Thus thefunctional properties of some native P_(2X) purinoceptors can beobtained by the expression of a single molecular species. The agonistinduced current recorded from ooctyes expressing the hP_(2X) clone givesa direct measure of the activation of P_(2X) -purinoceptors in a systemwith low levels of endogenous ectonucleotidase activity. The agonistprofile 2MeSATP≧ATP>α,β,-meATP for hP_(2X) is similar to that of thecloned rat vas deferens P_(2X) -purinoceptor. The high potency ofα,β,-meATP in whole tissue studies (α,β,-meATP >>2MeSATP≧ATP) probablyreflects, its resistance to ectonucleotidases.

The concentration-effect curves for ATP, 2MeSATP and 2-chloro-ATP weresuperimposable, indicating that these particular substitutions at the 2'position on the adenine ring do not affect agonist binding to the P_(2X)-purinoceptor. The agonist activity of AP₅ A is likely to be becausediadenosine phosphates (AP₅ A, and AP₆ A) released from the plateletscan act as vasoactive agents through activation of P_(2X)-purinoceptors.

Preferred features of each aspect of the invention are as for each otheraspect, mutatis mutandis.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be illustrated by the following examples. Theexamples refer to the accompanying drawings, in which:

FIGS. 1A-1C show DNA sequence (SEQ ID NO:4) and amino acid sequence (SEQID NO:5) of the rat vas deferens P_(2X) receptor as determined inExample 2.

FIGS. 2A-2C show DNA sequence (SEQ ID NO:6) and amino acid sequence (SEQID NO:7) of a rat superior cervical ganglion P_(2X) receptor, asdetermined in Example 11.

FIGS. 3A-3C show DNA sequence (SEQ ID NO:8) and amino acid sequence (SEQID NO:9) of a rat dorsal root ganglion P_(2X) receptor, as determined inExample 12.

FIGS. 4A-4D show DNA sequence (SEQ ID NO:10) and amino acid sequence(SEQ ID NO:11) of a human P_(2X) receptor as determined in Example 6.

FIG. 5 shows the alignment of the predicted amino acid sequence ofhP_(2X) (SEQ ID NO:11) with the rat vas deferens P_(2X) (SEQ ID NO:5),and in vitro translation of hP_(2X) protein.

TM1 and TM2 filled boxes indicate the hydrophobic regions and boxedamino acids indicate the differences between the two sequences,

o indicates conserved cysteine residues.

* Indicates potential sites of N-glycosylation.

FIG. 6 shows an SDS-PAGE analysis of ³⁵ S-methionine labelled hP_(2X)protein. Lanes 1 and 2 show in vitro coupled transcription/translationof pBKCMV-hP_(2X) cDNA in the absence and presence of microsomalmembranes, respectively.

FIGS. 7 AND 8 show Northern analyses of the hP_(2X) cDNA, wherein:

A) FIG. 7 shows Northern blot with 8 μg of total RNA from differentiatedHL60 cells.

0 indicates HL60 cells without treatment;

PMA2 and PMA3 indicate respectively cells treated 2 days, and 3 dayswith PMA;

DMSO indicates cells treated 6 days with DMSO;

dcAMP indicates cells treated 5 days with dibutryl cAMP;

UB indicates 100 ng of polyA⁺ RNA from human urinary bladder; and

B) FIG. 8 shows distribution of hP_(2X) in human tissues. Lanescontained 1 μg polyA⁺ RNA except for the urinary bladder which contained0.2 μg of polyA⁺ RNA.

FIGS. 9, 10 and 11 show the response of oocytes expressing hP_(2X) topurinoceptor agonists, wherein:

A) FIG. 9 shows traces which show inward currents evoked by ATP, 2 meSATP and α,β, me ATP (0.1, 1, and 100 μM). Records for each agonist arefrom separate oocytes;

B) FIG. 10 shows concentration response relationships of full P_(2X)-purinoceptor agonists. Data are expressed relative to the peak responseto 100 μM ATP; and

C) FIG. 11 shows concentration response of partial P_(2X) -purinoceptoragonists. Data are fitted with a Hill slope of 1 (n=4-8).

FIGS. 12 and 13 show the effects of P₂ -purinoceptor antagonists ofhP_(2X) mediated responses, wherein;

A) FIG. 12 shows concentration response curves for ATP in the presenceof the P₂ -purinoceptor agonist suramin (1, 10 and 100 μM) (n=4 for eachpoint); and

B) FIG. 13 shows concentration dependence of suramin, DIDS, PPADS and P₅P in inhibiting the response to 10 μM ATP (n=4 for each point).

FIGS. 14A-14D show the results of the functional characterisation of ratsuperior ganglion P_(2X) receptors (as encoded by clone 3, described inExample 10). These experiments provided electrical recordings fromtransfected HEK293 cells.

Top left: Superimposed currents evoked by ATP (30 μM) during the timeare indicated by the bar. Holding potential was changed from -70 to 20mV.

Top right: Peak current as a function of membrane potential.

Bottom left: Superimposed currents evoked by ATP, from 1 to 300 μM.

Bottom right: Concentration-response curves for ATP and αβmethylene-ATP(points are mean ±s.e. mean for 5-8 experiments).

FIG. 15A-15C shows the inhibition of currents caused by varioussubstances acting on the clone 3 form of the P_(2X) receptor (asdescribed in Example 11), compared with PC12 and human bladder forms inHEK293 cells.

Top: inhibition by suramin.

Middle: inhibition by PPADS.

Bottom: inhibition by pyridoxal 5-phosphate.

EXAMPLES (i) RAT VAS DEFERENS P_(2X) RECEPTOR EXAMPLE 1 Cloning of theRat vas deferens P_(2X) Receptor

Total RNA was isolated by the guanidinium isothiocyanate method(Sambrook et al., "Molecular Cloning: A Laboratory Manual" Cold SpringHarbor Laboratory Press, second edition (1989)) from vas deferens of 4weeks old Sprague-Dawley male rats, and the poly A+ RNA was subsequentlypurified by oligo(dT)-cellulose. First strand cDNA primed with thesequence 5'-GAGAGAGAGAGCGGCCGCTTTTTTTTTTTTTTT-3' (SEQ ID NO 1) wassynthesised with SUPERSCRIPT™ (BRL, Gaithersburg, Md., USA) Afterconversion of the cDNA to double stranded (Gubler & Hoffman, Gene 25263-269 (1983)) EcoRI linkers were ligated to the cDNA, and the productwas digested with NotI. The EcoRI-NotI cDNA of 1.3 to 9 kb was isolatedby gel electrophoresis, and a unidirectional library was constructed byligation of the cDNA to PBKCMV (Stratagene, San Diego, Calif., USA)digested with the same enzymes. The library was electroporated into E.coli DH10B cells and divided in 24 pools of 8×10⁴ clones. The plasmidDNA from the pools was prepared by minialkaline lysis followed by LiClprecipitation (Sambrook et al., loc. cit). NotI-linearised cDNA wastranscribed in vitro with T3 RNA polymerase in the presence of the capanalogue m7GpppG (Sambrook et al., loc. cit). The in vitro transcribedRNA (cRNA) was concentrated to 4 mg/ml.

EXAMPLE 2 Sequencing of the Rat vas deferens P_(2X) Receptor cDNA

The cDNA insert was sequenced the exonuclease method (Henikoff Meth.Enzymol. 155 156-164 (1987)). The sequence is shown in FIG. 1.

EXAMPLE 3 Functional characterisation of the Rat vas deferens P_(2X)Receptor cDNA in Oocytes

50 nl (200 ng) of RNA was injected into defolliculated Xenopus oocytes.After incubation for 2-6 days at 18° C., the oocytes were assayed forATP-evoked currents by a two-electrode voltage clamp (GENECLAMP™) ; oneelectrode is to hold the voltage constant (at -100 mV), and the other isto measure the currents. A cDNA pool which showed ATP induced currentswas subdivided to obtain a single clone (P_(2X)) Electrophysiologicalmeasurements were done at -100 mV, in a perfusion medium containing 96mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM Hepes pH 7.6, and 5 mMsodium pyruvate. For dose-response curves and suramin inhibition,oocytes were injected with 100 ng P_(2X) cRNA, and all recordings wereperformed at -60 mV, with Ba²⁺ substituted for external Ca²⁺ to preventactivation of endogenous Ca²⁺ -activated Cl⁻ currents. Microelectrodes(0.5-2 MΩ) were filled with 3M KCl.

EXAMPLE 4 Functional characterisation of the Rat vas deferens P_(2X)Receptor cDNA in HEX 293 Cells

HEK 293 cells were transfected by the lipofectin method (Felgner et al.,Proc. Nat'l. Acad. Sci. USA 84 7413-7417 (1987)) with P_(2X) -plasmid.DNA concentration used was 1 mg/2 ml medium placed into a 35 mm petridish containing four 11 mm diameter coverslips on which HEK cells wereplaced at 10,000 cells per coverslip. Cells were exposed tolipofectin/DNA for 6 h and recordings made 16-36 h later; 40-60% ofcells from which recordings were made exhibited P_(2X) responses.Currents were recorded from HEK 293 cells using whole-cell recordingmethods and the AXOPATCH™ 200 amplifier (Axon Instruments); patchpipettes (5 MΩ) contained (mM) Cs or K aspartate 140, NaCl 5, EGTA 11,HEPES 5. The external solution was (mM) NaCl 150, KCl2, CaCl₂ 2, MgCl₂1, HEPES 5 and glucose 11; the pH and osmolarity of both solutions weremaintained at 7.3 and 305 mosmol/l respectively. All recordingsperformed at room temperature. Data acquisition and analysis wereperformed using pCLAMP™ and AXOGRAPH™ software (Axon Instruments).Solutions for experiments examining calcium permeability of ATP currentsin HEK cells contained (mM) : internal solution NaCl 150, HEPES 5, CaCl₂0.5 and EGTA 5 (free calcium concentration about 5 nM); external sodiumsolution NaCl 150, glucose 11, histidine 5, CaCl₂ 2; external calciumsolution CaCl₂ 115, glucose 11 and histidine 5. The pH and osmolarity ofthe solutions were 7.4 and 295 mosmol/l respectively. For single channelmeasurements, a GENECLAMP™ 500 amplifier and outside-out recordingmethods were used (Adelman et al., Neuron 9 209-216 (1992)). Wax-coatedpatch pipettes (5-10 MΩ) contained (mM) K-gluconate 115, HEPES 5, BAPTA5 and MgCl₂ 0.5, external solution was 96 mM NaCl, 2 mM KCl, 1.8 mMCaCl₂, 1 mM MgCl₂, 5 mM Hepes pH 7.6, and 5 mM sodium pyruvate. ATP wasapplied by U-tube typically for 1 s; data was sampled at 5 kHz in 2 ssegments beginning 300 ms prior to onset of agonist (ATP) applicationand filtered at 1 kHz.

EXAMPLE 5 Transfection of the Rat vas deferens P_(2X) Receptor cDNA intoCHO and HEK293 Cells

CHO cells were stably transfected by a method used for other ionchannels (Claudio, Meth. Enzymol. 207 391-408 (1992)). Transfection wasconfirmed by a) electro-physiological recording and b) radioligandbinding. ATP and other agonists (up to 30 μM) caused rapidlydesensitising inward currents in 14 of 14 CHO cells stably transfected,and had no effect in 45 of 45 non-transfected cells. [³ H]αβmethyleneATPbinding was more than 600 cpm per million transfected cells with lessthan 80 cpm nonspecific binding.

Stable transfection of HEK293 cells was also achieved. This wasconfirmed by electrophysiological recording.

(ii) HUMAN P_(2X) RECEPTOR

The materials and methods used in the human P_(2X) receptor examples areset out below:

In Vitro translation

In vitro coupled transcription/translation were performed usingPromega's TNT Coupled reticulocyte lysate Systems with or without 2 μlof canine pancreatic microsomal membranes (Promega). μg CircularpBKCMV-hP_(2X) (0.5 ug) was transcribed with the T3 RNA polymerase asdescribed in the system manual in a 25 μl reaction for 2 h are 30° C.Synthesized proteins (5 μl) were analysed by SDS-PAGE andautoradiography.

Differentiation of HL60 cells

HL60 cells (human promyelocytes ATCC CCL240) were passaged twice weeklyin RPMI-1640 supplemented with 25 mnM HEPES, 2 mM Glutamax II, and 10%heat-inactivated fetal calf serum (GIBCO BRL) . For each experiment33×10⁶ cells were resuspended at 2.5×10⁵ cells/ml in medium containingeither phorbol mystate acetate (100 nM), 1. 1% DMSO, or dibutyryl cAMP(200 uM) (SIGMA) for the indicated times.

Northern blot analysis

PolyA⁺ RNAs were obtained from Clontech Laboratories Inc. (Palo Alto)except for the urinary bladder and HL60 mRNA which were prepared asdescribed (Valera et al (1994)--supra) . Samples were quantified bymeasuring the O.D. at 260 nm, and by staining the membrane withmethylene blue. The RNA were fractionated on a 1% agarose--6%formaldehyde gel and electroblotted to a non-charged nylon membrane(BDH). Prehybridisation at 68° C. was performed for 6 hours inhybridisation buffer (50% formamide, 5× SSC, 2% blocking buffer(Boehringer Mannheim), 0.1%6 laurolylsarcosine, 0.02% SDS).Hybridisation was overnight at 68° C. in fresh hybridisation buffer witha digoxigenin-UTP labelled riboprobe (100 ng/ml) corresponding to theentire hP₂ × sequence. The membrane was washed at 68° C.; twice in 2×SSC+0.1% SDS, and twice in 0.1× SSC+0.1% SDS. Chemiluminescent detectionof hybridisation was carried at room temperature as follows: themembrane was rinsed 5 min in buffer B1 (0.1 M maleic acid, 0.15 M NaCl,pH 7.5), saturated for 1 hour in 1% blocking buffer (B2), incubated 30min with anti-digoxigenin-antibody alkaline phosphatase conjugated (750u/ml, Boehringer Mannheim) diluted 1:15000 in B2, washed in B1+0.30%tween 20 (1× 5 min, 1× 15 min, 1× 1 h), equilibrated for 5 min in bufferB3 (0.1 M Tris HCl pH 9.5, 0.1 M NaCl, 50 mM MgCl₂), incubated 45-60 secin lumigen PPD (Boehringer Mannheim) diluted 1:100 in B3. The humidmembrane was sealed in a plastic bag, incubated 15 min at 37° C., andexposed 15 to 20 min to Hyperfilm-ECL (Amersham).

P_(2X) expression into oocytes

Human urinary bladder P_(2X) cDNA, subcloned into the pBKCMV expressionvector, was linearized with Notl, and transcribed in vitro with T3polymerase in the presence of cap analogue m7G(5')ppp(5')G.Defolliculated Xenopus oocytes (Bertrand et al, Electrophysiology ofneuronal nicotinic acetylcholine receptors expressed in Xenopus oocytesfollowing nuclear injection of genes or cDNAs Meth Neurosci 4: 174-193(1991)) were injected with 50 ng of human P_(2X) in vitro transcribedRNA, and incubated at 18° C. for 2-6 days in the ND96 solution (mM):NaCl96, KCl2, MgCl₂ 1, CaCl₂ 2, sodium pyruvate 5, HEPES 5, ph 7.6-7.5,penicillin (10 U/ml), and streptomycin (10 μg/ml).

Electrophysiology

Oocytes were placed in a 1 ml chamber and superfused at 2-3 ml/min withND96 solution with 0.1 mM BaCl₂ replacing the 2 mM CaCl₂ to preventactivation of endogenous calcium-activated chloride currents (Barish, Atransient calcium-dependent chloride current in the immature Xenopusoocytes J Physiol 342: 309-325 (1983)). Currents were measured using atwo-electrode voltage-clamp amplifier (Geneclamp Axon Instruments) at aholding potential of -60 mV. Microelectrodes were filled with 3 M KCl(0.5-2 MΩ). Data were collected using PClamp software (AxonInstruments). ATP and other purinoceptor agonists were applied by aU-tube perfusion system (Fenwick et al, A patch clamp study of bovinechromaffin cells and their sensitivity to acetylcholine J Physiol 331:577-597 (1982)) placed close (200-500 μm) to the oocyte. Initial studiesshowed that reproducible responses (<10% variation in peak amplitude)could be obtained when ATP (at concentrations up to 1 mM) was applied tohP_(2X) injected oocytes for 5 s every 10 mins. Concentration responserelationships to ATP and its analogs were determined by measuring thepeak amplitude of responses to a 5 s application of agonist applied at10 min intervals. Responses to agonists were normalized in each oocyteto the peak response evoked by 100 μM ATP; 100 μM ATP was usuallyapplied at the beginning and at the end of an experiment to determine ifthere was any rundown of the response. No inward current was recorded inuninjected oocytes in response to application of purinoceptor agonistsat the maximal concentration used (n=3 for each agonist). Antagonistswere applied both in the superfusate and together with ATP in the U-tubesolution. Antagonists were superfused for 5-10 min prior to theapplication of ATP.

Data analysis

Concentration response curves for purinoceptor agonists were fitted witha Hill slope of 1. Equi-effective concentrations i.e. concentration ofagonist, giving 50% of the response to 100 μM ATP, (EEC₅₀) weredetermined from individual concentration response curves. Forantagonists the concentration required to give 50% inhibition (IC50) ofthe response to 10 μM ATP (approximately 90% of peak response to ATP)were determined. Data are presented throughout as mean ± SEM for a givennumber of oocytes.

Drugs

Adenosine, adenosine 5'-monophosphate sodium salt (AMP), adenosine5'-diphosphate sodium salt (ADP), adenosine 5'-triphosphate magnesiumsalt (ATP), adenosine 5'-O-(-3-thiophosphate) tetralithium salt(ATP-γ-S), uridine 5'-triphosphate sodium salt (UTP), α,β-methylene ATPlithium salt (α,β,-meATP), β,γ-methylene-D-ATP sodium salt(D-β,γ-meATP), 2'-3'-O-(4-benzoylbenzol)ATP tetraethylamonium salt(BzATP), 4,4'-diisothiocyanatostilbene 2,2'-disulphonic acid, disodiumsalt (DIDS) were obtained from Sigma. 2-MethylthioATP tetra sodium salt(2MeSATP), 2-chloro-ATP tetra sodium salt, and β-γ-methylene-1-ATP(1-β-γ-meATP) were obtained from RBl. Pyridoxal 5-phosphate monohydrate(Aldrich), p1, p5-di [adenosine-5'] pentaphosphate trilithium salt(AP5A) (Boehringer Mannheim), pyridoxal phosphate 6-azophenyl2',4'-disulphonic acid (PPADS, gift of G. Lambrecht, University ofFrankfurt) and suramin (Bayer) were tested. Drugs were prepared fromfrozen aliquots of stock solutions and diluted to give the requiredfinal concentration.

EXAMPLE 6 Sequence and characteristics of hP_(2X) from urinary bladder

Isolation of human P_(2X) cDNA

Human urinary bladder tissue was obtained from a cystectomy for abladder tumor. The patient showed no symptoms of bladder instability orurodynamic abnormalities. Only those portions, surrounding the tumor,which appeared macroscopically normal (Palea et al--supra) were used.Total RNA was isolated by guanidinium isothiocyanate and poly A⁺ RNA waspurified as described (Valera et al (1994)--supra). Preparation of acDNA library in λgt10, random primer labelling of a rat smooth muscleP_(2X) probe (Valera et al (1994)--supra), low stringency hybridisationscreening and lambda phage DNA isolation were all done by standardprotocols (Sambrook et al, Molecular Cloning, A Laboratory Manual, 2ndedn., Cold Spring Harbor Laboratory Press, New York (1989)). Severalindependent phage isolates were examined and the cDNA insert from onewas chosen for subcloning into Eco RI-Not I digested pBKCMV. This 2677bp hP_(2X) cDNA was sequenced as described (Valera et al (1994)--supra).

The 2677 bp cDNA, hP_(2X), contained a single long open reading framewhich corresponds to a protein of 399 amino acids (FIG. 4). This aminoacid sequence is highly homologous with that of the P_(2X) receptor,isolated from rat vas deferens (89% identity). There are two regions ofhydrophobicity near either end of the protein which are sufficientlylong to traverse the membrane but there is no hydrophobic N-terminalleader sequence. All five potential sites for glycosylation and all tencysteine residues in the central section of the protein are conserved.In vitro translation of hP_(2X) RNA in the presence of microsomesproduced a 60 kD product, whereas translation in the absence ofmicrosomes produced the 45 kD peptide (FIG. 6). 45 kD is the computedmolecular weight, suggesting that the additional 15 kD results fromglycosylation.

Some human urinary bladder P_(2X) cDNA was used to transfect HEK293cells. Stable transfection was confirmed by electrophysiologicalrecording.

EXAMPLE 7 Distribution of human urinary bladder P_(2X) mRNA

The distribution of the human urinary bladder P_(2X) mRNA was examinedby northern analysis. A single 2.6 kb mRNA species was observed inbladder, placenta, liver and adrenal gland (FIG. 8). In thymus, spleen,and lung samples, the 2.6 kb band plus additional higher molecularweight RNAs of 3.6 and 4.2 kb were seen. A smaller additional RNAspecies of 1.8 kb was observed in spleen and lung. No hybridisation wasdetected with brain mRNA.

EXAMPLE 8 Induction of hP_(2X) mRNA in HL60 cells

A portion of the 3'-untranslated region had been previously deposited inthe database (HSGS01701) as an expressed sequence tag for thedifferentiation of the human promyelocytic cell line, HL60 (Okubounpublished). We examined the induction of hP_(2X) mRNA in HL60 cells byNorthern blot analysis (FIG. 7). HL60 cells can be differentiated intodistinct lineages, depending on the inductant (Koeffler, Induction ofDifferentiation of Human Acute Myelogenous Leukemia Cells: TherapeuticImplications Blood 62: 709-721 (1983)). Induction of macrophage-likecharacteristics with phorbol diesters or granulocytic differentiationwith DMSO or dibutryl cAMP, each produced an increase in P_(2X) mRNA(FIG. 7, lane 6), HL60 RNA (lane 1-5) showed hybridisation of two bands(1.8 and 2.6 kb) and both of these were inducible. This contrasts withthe bladder, where Northern analysis showed only a single RNA species(2.6 kb) (FIG. 7, lane 6).

EXAMPLE 9 Pharmacological characterization of hP_(2X)

Application of ATP (30 nM-1 mM) to oocytes injected with hP_(2X)receptor RNA evoked inward currents (FIGS. 9, 10 and 11). Responses tolow concentrations of ATP (30-300 nM) developed over 3-5 s. Higherconcentrations of ATP (1 μM) evoked responses which peaked within 1-1.5s and then declined during the continued application of ATP (40-60Cr ofthe peak amplitude after 5 s). The current returned to control values onwashout of ATP. The peak amplitude of the inward current evoked by ATPwas concentration-dependent (FIGS. 9, 10 and 11) and could be fitted bya curve with a Hill slope of 1 with a EC₅₀ of 0.82 μM. When ATP (100 μM)was applied for 5 s every 10 min, reproducible inward currents wererecorded. This is in contrast to the responses of the P_(2X) receptorclone from rat vas deferens where a second application of ATP (>1 μM)applied 10 mins after the first, evoked an inward current that was ˜50%of the initial peak amplitude.

Concentration-response curves were constructed for a number of other P₂purinoceptor agonists (FIGS. 9, 10 and 11). 2meSATP, 2-chloro-ATP,α,β,-meATP and ADP were full agonists. BZATP, AP₅ A and ATP-γ-S producedmaximal responses of about 65% of the maximal ATP response. The maximalresponses to d and 1-β,γ-meATP were not determined. Adenosine, AMP andUTP (100 μM) evoked small inward currents (2.3±1.5, 6.08±2, and 3.7±1.8%of the response to 100 μM ATP respectively). The EECD₅₀ values andrelative potencies of purinoceptor analogs are summarised in Table 1below.

                  TABLE 1                                                         ______________________________________                                        agonist       EEC50 (μM)                                                                           relative potency                                      ______________________________________                                        ATP           0.82      1                                                       2MeSATP 0.6 ± 0.1 1.36                                                     2chloroATP 0.76 ± 0.1  1.08                                                AP5A   2 ± 0.2 0.41                                                        α,β-meATP 3.6 ± 1.6 0.23                                        BzATP 4.2 ± 2.2 0.20                                                       ATP-γ-S 10.6 ± 3.8  0.077                                            d,β,γ-meATP 24.1 ± 1.6  0.034                                   ADP 34.3 ± 16   0.024                                                    ______________________________________                                         EEC50: Equieffective concentrations producing an inward current equivalen     to 50% of the peak response to 100 μM ATP. EEC50 taken from individual     fitted concentration response curves with a Hill slope of 1. EEC50 for AT     from mean data from all experiments. (n = 3-4).                          

EXAMPLE 10 Antagonist studies

The P₂ -purinoceptor antagonist suramin (1-100 μM) shifted theconcentration-response curve for ATP to the right. At 1 μM suramin theshift was almost parallel. The dissociation equilibrium constant (K_(B))estimated from K_(B) =1/(DR-1) where DR is the dose ratio was 130 nM.With higher concentrations of suramin the inhibition did not appear tobe competitive. Under the present experimental conditions this K_(B)estimate is higher than those reported previously for suramin (pA2 5.9,Trezise et al, Br J Pharmacol 112: 282-288 (1994)) pK_(B) 5.2, vonKugelgen et al, Interaction of adenine nucleotides, UTP and suramin inmouse vas deferens: suramin-sensitive and suramin-insensitive componentsin the contractile effect of ATP Naunyn Schmiedeberg's Arch Pharmacol342: 198-205 (1990)). The antagonism by suramin was fully reversed after10 mins wash and indicates that the non-competitive antagonism at highconcentrations is not due to irreversible binding of the antagonist tothe receptor.

The putative P_(2X) purinoceptor antagonists PPADS, DIDS and pyridoxal 5phosphate (Ziganshin et al, Selective antagonism by PPADS at P_(2X)purinoceptors in rabbit isolated blood vessels Br J Pharmacol 111:923-929 (1994), Bultmann & Starke, Blockade by4,4'-diisothiocyanatostilben-2,2'-disulphonate (DIDS) of P_(2X)purinoceptors in rat vas deferens Br J Pharmacol 112: 690-694 (1994),Trezise et al, Eur J Pharmacol 259: 295-300 (1994)) inhibited inwardcurrents evoked by 10 μM ATP (approximately EC₉₀ concentration) in aconcentration dependent manner (FIGS. 12 and 13). Suramin PPADS and DIDSwere equally effective in inhibiting ATP evoked currents (IC₅₀ ˜1 μM).The IC 50 for P₅ P was˜20 μM. PPADS and P₅ P antagonism was readilyreversible on washout. In contrast, inhibitory effects of DIDS (100 μM)were very slow to reverse on washout.

(iii) RAT SUPERIOR CERVICAL GANGLION P_(2X) RECEPTOR EXAMPLE 11Isolation and functional expression of a cDNA encoding a P_(2X) receptorfrom rat superior cervical ganglion (referred to herein as clone 3)

A 440 bp fragment was amplified by polymerase chainreaction (PCR) fromrat testis cDNA, using degenerate primers based on conserved nucleotidesequences within the rat vas deferens P_(2X) receptor cDNA and on thesequence of PC12 cDNA (Ehrlich H A (ed) PCR Technology MacMillan,Basingstoke (1989)). The primers used are given below: Sense(SEQ ID NO2) - 5' T G T/C G A A/G A/G T I T T/C I G G/C I T G G T G T/C C C 3' -Antisense(SEQ ID NO 3) - 5' G C A/G A A T/C C T A/G A A A/G T T A/G T/AA I C C 3'

(wherein I=Inosine and "T/C" indicates that either T or C is present atthe position indicated (this applies mutatis mutandis to the otheralternatives given).

The cloned PCR fragment was labelled and used as a hybridization probefor screening a rat testis cDNA bank in λZAP. One recombinant phage waspositive, and its insert was excised and transferred to a plasmid(#432). This cDNA was 1500 bp with a single EcoR1 site (at position1000, still in the open reading frame). The 5' end of the cDNA was tooshort to encode the entire N terminus.

Internal primers specific to the new sequence were made and the tissuedistribution was tested by PCR. The candidate was present in mRNAprepared from phaeochromocytoma (PC12) cells, intestine and superiorcervical ganglion (scg). The hybridization probe was therefore used toscreen a rat scg cDNA bank in λgt10. From 30 initial positives, 20 purephage DNA stocks were prepared; 19 were various portions of thecandidate sequence, and the insert from one was transferred to plasmid(p457) and sequenced. The insert appeared to be a full length cDNA; ithas a single open reading frame of 388 amino acids (FIG. 2). The insertfrom p457 was subcloned into pcDNA3 (p464) and used to transfect humanembryonic kidney (HEK293) cells.

The functional characterisation of the clone illustrated in FIG. 2(referred to herein as clone 3) was carried out by electrical recordingsfrom transfected HEK293 cells and from oocytes injected with the invitro transcribed RNA, as described in Example 4 for the rat vasdeferens P_(2X) receptor. Table A summarizes the main properties ofclone 3 as compared to those of rat vas/human bladder cDNA clone, andthe PC12 cDNA clone (provided by David Julius and Tony Brake of theUniversity of California at San Francisco).

                  TABLE A                                                         ______________________________________                                        Functional Properties of 3 cloned P.sub.2x Receptors                                    bladder     clone 3     PC12                                        ______________________________________                                        kinetics                                                                        desensitization very strong very little very little                           rundown profound very little very little                                      ionic permeability                                                            monovalent no differences no differences no differences                       divalent (Ca.sup.++) high permeability high permeability high                 Ca.sup.++  block none intermediate permeability                                  very strong                                                                agonist profile                                                               ATP 0.7 μM 11 μM 8 μM                                                α,β-meATP 3 μM >>100 mM >>100 μM                             antagonist profile                                                            suramin 1 μM <40% block 6 μM                                            PPADS 1 μM <30% block 1 μM                                              P-5-P 6 μM <40% block 6 μM                                              DIDS 1 μM  >100 μM                                                    ______________________________________                                    

The main functional properties of clone 3 are as follows. (a) Thecurrents evoked by ATP show little or no decline during applications ofseveral seconds; that is, there is little desensitisation (FIG. 14). (b)The relative permeabilities of the ionic pore to sodium, potassium,cesium, tetraethylammonium and to calcium are not different to thoseobserved for the rat vas deferens/human bladder or the PC12 forms of thereceptor. (c) Extracellular calcium (30 mM) inhibits the inward currentthrough the P_(2X) receptor channel of the PC12 form whereas it does notblock current through the rat vas deferens/human bladder form; clone 3is intermediate in sensitivity. (d) The effectiveness of agonists thatare structurally related to ATP is the same as that found for the PC12form; most notably, αβmethylene ATP has little or no agonist action(FIG. 14). (e) Currents activated by ATP at the clone 3 receptor weremuch less sensitive to antagonism by suramin., pyridoxal 5'-phosphateand pyridoxal-6-azophenyl-2',4'-disulphonic acid (PPADS) than weresimilar current mediated by the other two forms (rat vas deferens/humanbladder; PC12) (FIG. 15).

(iv) RAT DORSAL ROOT GANGLION P_(2X) RECEPTOR EXAMPLE 12 Isolation of acDNA encoding a P_(2X) receptor from a rat dorsal root ganglion

By using PCR with the same primers as used in Example 11 above, butusing different cDNA sources, further P_(2X) family members can befound.

Using this method, rat dorsal root ganglion P_(2X) receptor cDNA wasisolated. FIG. 1B shows the cDNA sequence of this clone (referred toherein as clone 6), together with the putative amino acid sequence. Theportions underlined in this figure correspond to the PCR primersinitially used.

A similar procedure to that described in Example 11 was then used toisolate the full length cDNA.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 11                                          - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - GAGAGAGAGA GCGGCCGCTT TTTTTTTTTT TTT       - #                  -      #         33                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - TGNGANNTNT NNGNNTGGTG NCC           - #                  - #                    23                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - GCNAANCTNA ANTTNNANCC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1837 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (vii) IMMEDIATE SOURCE:                                                         (B) CLONE: rat P2x f - #rom vas deferens                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - GCCAAAAGCT GTTCTGATCA CCCAGGGTTT TTCCTCCCAA CCCAGACCCC AC -             #CATCGAAC     60                                                                 - - CTCCAACTCT GGTCCCACCT AGCCTGCTCT GTCCTTAAGG GGCCGGGAAG CC -            #CCAGTCAC    120                                                                 - - TCCACTGCTA TTGTAGATGC AGATGGTGGC CTGCCCTTGA CCATAGAGGC CG -            #TGTGGGGT    180                                                                 - - GTTCATCTCT GAGCCCCTTC TGGCCCACC ATG GCT CGG CGG CTG - # CAA GAT        GAG      233                                                                                      - #              Met Ala A - #rg Arg Leu Gln Asp Glu                         - #                1  - #             5                      - - CTG TCA GCC TTC TTC TTT GAA TAT GAC ACT CC - #C CGG ATG GTG CTG GTA          281                                                                       Leu Ser Ala Phe Phe Phe Glu Tyr Asp Thr Pr - #o Arg Met Val Leu Val                10             - #     15             - #     20                          - - CGA AAC AAG AAG GTG GGA GTC ATT TTC CGT CT - #G ATC CAG TTG GTG GTT          329                                                                       Arg Asn Lys Lys Val Gly Val Ile Phe Arg Le - #u Ile Gln Leu Val Val            25                 - # 30                 - # 35                 - # 40       - - CTG GTC TAC GTC ATT GGG TGG GTG TTT GTC TA - #T GAA AAA GGA TAC CAG          377                                                                       Leu Val Tyr Val Ile Gly Trp Val Phe Val Ty - #r Glu Lys Gly Tyr Gln                            45 - #                 50 - #                 55              - - ACC TCA AGT GAC CTC ATC AGC AGT GTG TCC GT - #G AAG CTC AAG GGC TTG          425                                                                       Thr Ser Ser Asp Leu Ile Ser Ser Val Ser Va - #l Lys Leu Lys Gly Leu                        60     - #             65     - #             70                  - - GCT GTG ACC CAG CTC CAG GGC CTG GGA CCC CA - #G GTC TGG GAC GTG GCT          473                                                                       Ala Val Thr Gln Leu Gln Gly Leu Gly Pro Gl - #n Val Trp Asp Val Ala                    75         - #         80         - #         85                      - - GAC TAT GTC TTC CCA GCA CAC GGG GAC AGC TC - #C TTT GTA GTT ATG ACC          521                                                                       Asp Tyr Val Phe Pro Ala His Gly Asp Ser Se - #r Phe Val Val Met Thr                90             - #     95             - #    100                          - - AAC TTC ATC GTG ACC CCT CAG CAG ACT CAA GG - #C CAT TGT GCA GAG AAC          569                                                                       Asn Phe Ile Val Thr Pro Gln Gln Thr Gln Gl - #y His Cys Ala Glu Asn           105                 1 - #10                 1 - #15                 1 -      #20                                                                              - - CCA GAA GGT GGC ATA TGC CAG GAT GAC AGT GG - #C TGC ACT CCA GGA        AAA      617                                                                    Pro Glu Gly Gly Ile Cys Gln Asp Asp Ser Gl - #y Cys Thr Pro Gly Lys                          125  - #               130  - #               135              - - GCA GAA AGG AAA GCC CAA GGT ATT CGC ACA GG - #C AAC TGT GTG CCC TTC          665                                                                       Ala Glu Arg Lys Ala Gln Gly Ile Arg Thr Gl - #y Asn Cys Val Pro Phe                       140      - #           145      - #           150                  - - AAT GGC ACT GTG AAG ACA TGT GAG ATC TTT GG - #T TGG TGT CCT GTA GAG          713                                                                       Asn Gly Thr Val Lys Thr Cys Glu Ile Phe Gl - #y Trp Cys Pro Val Glu                   155          - #       160          - #       165                      - - GTG GAT GAC AAG ATC CCA AGC CCT GCT CTT CT - #T CGT GAG GCT GAG AAC          761                                                                       Val Asp Asp Lys Ile Pro Ser Pro Ala Leu Le - #u Arg Glu Ala Glu Asn               170              - #   175              - #   180                          - - TTC ACC CTC TTC ATC AAA AAC AGC ATC AGC TT - #T CCA CGC TTC AAG GTC          809                                                                       Phe Thr Leu Phe Ile Lys Asn Ser Ile Ser Ph - #e Pro Arg Phe Lys Val           185                 1 - #90                 1 - #95                 2 -      #00                                                                              - - AAC AGG CGC AAC CTG GTA GAG GAG GTG AAC GG - #C ACC TAC ATG AAG        AAG      857                                                                    Asn Arg Arg Asn Leu Val Glu Glu Val Asn Gl - #y Thr Tyr Met Lys Lys                          205  - #               210  - #               215              - - TGC CTC TAT CAC AAG ATT CAA CAC CCC CTG TG - #C CCA GTC TTC AAC CTT          905                                                                       Cys Leu Tyr His Lys Ile Gln His Pro Leu Cy - #s Pro Val Phe Asn Leu                       220      - #           225      - #           230                  - - GGC TAT GTG GTG CGA GAG TCA GGC CAG GAC TT - #C CGC AGC CTT GCT GAG          953                                                                       Gly Tyr Val Val Arg Glu Ser Gly Gln Asp Ph - #e Arg Ser Leu Ala Glu                   235          - #       240          - #       245                      - - AAG GGT GGG GTG GTT GGT ATC ACC ATT GAC TG - #G AAG TGT GAT CTG GAC         1001                                                                       Lys Gly Gly Val Val Gly Ile Thr Ile Asp Tr - #p Lys Cys Asp Leu Asp               250              - #   255              - #   260                          - - TGG CAC GTT CGG CAC TGC AAA CCC ATC TAC CA - #G TTC CAC GGA CTG TAT         1049                                                                       Trp His Val Arg His Cys Lys Pro Ile Tyr Gl - #n Phe His Gly Leu Tyr           265                 2 - #70                 2 - #75                 2 -      #80                                                                              - - GGG GAG AAG AAC CTG TCT CCA GGC TTC AAC TT - #C AGA TTT GCC AGG        CAT     1097                                                                    Gly Glu Lys Asn Leu Ser Pro Gly Phe Asn Ph - #e Arg Phe Ala Arg His                          285  - #               290  - #               295              - - TTC GTG CAG AAT GGG ACA AAC CGT CGT CAC CT - #C TTC AAG GTG TTT GGG         1145                                                                       Phe Val Gln Asn Gly Thr Asn Arg Arg His Le - #u Phe Lys Val Phe Gly                       300      - #           305      - #           310                  - - ATT CAC TTT GAT ATC CTT GTG GAT GGC AAG GC - #T GGG AAG TTT GAC ATC         1193                                                                       Ile His Phe Asp Ile Leu Val Asp Gly Lys Al - #a Gly Lys Phe Asp Ile                   315          - #       320          - #       325                      - - ATC CCT ACT ATG ACT ACT ATC GGT TCT GGG AT - #T GGC ATC TTT GGA GTG         1241                                                                       Ile Pro Thr Met Thr Thr Ile Gly Ser Gly Il - #e Gly Ile Phe Gly Val               330              - #   335              - #   340                          - - GCC ACA GTG CTT TGT GAT CTC TTA TTG CTC CA - #C ATC CTG CCT AAG AGG         1289                                                                       Ala Thr Val Leu Cys Asp Leu Leu Leu Leu Hi - #s Ile Leu Pro Lys Arg           345                 3 - #50                 3 - #55                 3 -      #60                                                                              - - CAC TAC TAC AAG CAG AAG AAG TTC AAA TAT GC - #C GAG GAC ATG GGG        CCG     1337                                                                    His Tyr Tyr Lys Gln Lys Lys Phe Lys Tyr Al - #a Glu Asp Met Gly Pro                          365  - #               370  - #               375              - - GGA GAG GGT GAA CAT GAC CCC GTG GCC ACC AG - #C TCC ACT CTG GGC CTG         1385                                                                       Gly Glu Gly Glu His Asp Pro Val Ala Thr Se - #r Ser Thr Leu Gly Leu                       380      - #           385      - #           390                  - - CAG GAG AAC ATG AGG ACC TCC TGACCTTAGT CTTGAGATC - #C GGACTTGACG            1436                                                                       Gln Glu Asn Met Arg Thr Ser                                                           395                                                                    - - CAGTGTGTGG CTTCCGGCAA GGGCTGATGG CTTTGAGCCA GGGCAGAGGG CA -             #TTCCCAGA   1496                                                                 - - GGCTTTCCTG CAAGGCAGAC ACCAGTGGCC CTCTGGTTCA GCATGAAGAC AG -            #GCAAGACT   1556                                                                 - - TTGGATTTCA GAGCTCTGGT TTCAGTTCCA CATGTCCCTT CCTGAGGGAT GC -            #CTCCTCCA   1616                                                                 - - GTTTTCACCA ATTTGGGTTC ATATGGCTGG GCCCCTCACA CATCTATACT CT -            #AGCTTTGT   1676                                                                 - - GCTTAAGGCT CAGGCTGTCA TTGTCTTTCC CACAGCCTTA CCTGCCTAGA TT -            #TGGGCTCT   1736                                                                 - - TCCACATGGT AGCCACTAGC CAGATGTGTC AGTTTGAACT TTAATTAAAA TA -            #TAATAAAA   1796                                                                 - - AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA A    - #                      - # 1837                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 399 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - - Met Ala Arg Arg Leu Gln Asp Glu Leu Ser Al - #a Phe Phe Phe Glu Tyr        1               5 - #                 10 - #                 15              - - Asp Thr Pro Arg Met Val Leu Val Arg Asn Ly - #s Lys Val Gly Val Ile                   20     - #             25     - #             30                  - - Phe Arg Leu Ile Gln Leu Val Val Leu Val Ty - #r Val Ile Gly Trp Val               35         - #         40         - #         45                      - - Phe Val Tyr Glu Lys Gly Tyr Gln Thr Ser Se - #r Asp Leu Ile Ser Ser           50             - #     55             - #     60                          - - Val Ser Val Lys Leu Lys Gly Leu Ala Val Th - #r Gln Leu Gln Gly Leu       65                 - # 70                 - # 75                 - # 80       - - Gly Pro Gln Val Trp Asp Val Ala Asp Tyr Va - #l Phe Pro Ala His Gly                       85 - #                 90 - #                 95              - - Asp Ser Ser Phe Val Val Met Thr Asn Phe Il - #e Val Thr Pro Gln Gln                  100      - #           105      - #           110                  - - Thr Gln Gly His Cys Ala Glu Asn Pro Glu Gl - #y Gly Ile Cys Gln Asp              115          - #       120          - #       125                      - - Asp Ser Gly Cys Thr Pro Gly Lys Ala Glu Ar - #g Lys Ala Gln Gly Ile          130              - #   135              - #   140                          - - Arg Thr Gly Asn Cys Val Pro Phe Asn Gly Th - #r Val Lys Thr Cys Glu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ile Phe Gly Trp Cys Pro Val Glu Val Asp As - #p Lys Ile Pro Ser        Pro                                                                                             165  - #               170  - #               175             - - Ala Leu Leu Arg Glu Ala Glu Asn Phe Thr Le - #u Phe Ile Lys Asn Ser                  180      - #           185      - #           190                  - - Ile Ser Phe Pro Arg Phe Lys Val Asn Arg Ar - #g Asn Leu Val Glu Glu              195          - #       200          - #       205                      - - Val Asn Gly Thr Tyr Met Lys Lys Cys Leu Ty - #r His Lys Ile Gln His          210              - #   215              - #   220                          - - Pro Leu Cys Pro Val Phe Asn Leu Gly Tyr Va - #l Val Arg Glu Ser Gly      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gln Asp Phe Arg Ser Leu Ala Glu Lys Gly Gl - #y Val Val Gly Ile        Thr                                                                                             245  - #               250  - #               255             - - Ile Asp Trp Lys Cys Asp Leu Asp Trp His Va - #l Arg His Cys Lys Pro                  260      - #           265      - #           270                  - - Ile Tyr Gln Phe His Gly Leu Tyr Gly Glu Ly - #s Asn Leu Ser Pro Gly              275          - #       280          - #       285                      - - Phe Asn Phe Arg Phe Ala Arg His Phe Val Gl - #n Asn Gly Thr Asn Arg          290              - #   295              - #   300                          - - Arg His Leu Phe Lys Val Phe Gly Ile His Ph - #e Asp Ile Leu Val Asp      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Gly Lys Ala Gly Lys Phe Asp Ile Ile Pro Th - #r Met Thr Thr Ile        Gly                                                                                             325  - #               330  - #               335             - - Ser Gly Ile Gly Ile Phe Gly Val Ala Thr Va - #l Leu Cys Asp Leu Leu                  340      - #           345      - #           350                  - - Leu Leu His Ile Leu Pro Lys Arg His Tyr Ty - #r Lys Gln Lys Lys Phe              355          - #       360          - #       365                      - - Lys Tyr Ala Glu Asp Met Gly Pro Gly Glu Gl - #y Glu His Asp Pro Val          370              - #   375              - #   380                          - - Ala Thr Ser Ser Thr Leu Gly Leu Gln Glu As - #n Met Arg Thr Ser          385                 3 - #90                 3 - #95                            - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1997 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                           - - CGCAGCGAGC CTGCCGGAGC TGGTGGGTGG AGCTACGACC GGGAGCCGAC GG -             #TGGCGAGG     60                                                                 - - GGACCCACAG TGTCCAAGGC GCGGAGCGGT CGGCGGAGCC ATG GCG GG - #C TGC        TGC      115                                                                                      - #                  - #        Met Ala Gly Cys Cys                          - #                  - #        400                          - - TCC GTG CTC GGG TCC TTC CTG TTC GAG TAC GA - #C ACG CCG CGC ATC GTG          163                                                                       Ser Val Leu Gly Ser Phe Leu Phe Glu Tyr As - #p Thr Pro Arg Ile Val           405                 4 - #10                 4 - #15                 4 -      #20                                                                              - - CTC ATC CGC AGC CGT AAA GTG GGG CTC ATG AA - #C CGC GCG GTG CAG        CTG      211                                                                    Leu Ile Arg Ser Arg Lys Val Gly Leu Met As - #n Arg Ala Val Gln Leu                          425  - #               430  - #               435              - - CTC ATC CTG GCT TAC GTC ATC GGG TGG GTG TT - #C GTG TGG GAA AAG GGC          259                                                                       Leu Ile Leu Ala Tyr Val Ile Gly Trp Val Ph - #e Val Trp Glu Lys Gly                       440      - #           445      - #           450                  - - TAC CAG GAA ACG GAC TCC GTG GTC AGC TCG GT - #G ACA ACC AAA GCC AAA          307                                                                       Tyr Gln Glu Thr Asp Ser Val Val Ser Ser Va - #l Thr Thr Lys Ala Lys                   455          - #       460          - #       465                      - - GGT GTG GCT GTG ACC AAC ACC TCT CAG CTT GG - #A TTC CGG ATC TGG GAC          355                                                                       Gly Val Ala Val Thr Asn Thr Ser Gln Leu Gl - #y Phe Arg Ile Trp Asp               470              - #   475              - #   480                          - - GTG GCG GAC TAT GTG ATT CCA GCT CAG GAG GA - #A AAC TCC CTC TTC ATT          403                                                                       Val Ala Asp Tyr Val Ile Pro Ala Gln Glu Gl - #u Asn Ser Leu Phe Ile           485                 4 - #90                 4 - #95                 5 -      #00                                                                              - - ATG ACC AAC ATG ATT GTC ACC GTG AAC CAG AC - #A CAG AGC ACC TGT        CCA      451                                                                    Met Thr Asn Met Ile Val Thr Val Asn Gln Th - #r Gln Ser Thr Cys Pro                          505  - #               510  - #               515              - - GAG ATT CCT GAT AAG ACC AGC ATT TGT AAT TC - #A GAC GCC GAC TGC ACT          499                                                                       Glu Ile Pro Asp Lys Thr Ser Ile Cys Asn Se - #r Asp Ala Asp Cys Thr                       520      - #           525      - #           530                  - - CCT GGC TCC GTG GAC ACC CAC AGC AGT GGA GT - #T GCG ACT GGA AGA TGT          547                                                                       Pro Gly Ser Val Asp Thr His Ser Ser Gly Va - #l Ala Thr Gly Arg Cys                   535          - #       540          - #       545                      - - GTT CCT TTC AAT GAG TCT GTG AAG ACC TGT GA - #G GTG GCT GCA TGG TGC          595                                                                       Val Pro Phe Asn Glu Ser Val Lys Thr Cys Gl - #u Val Ala Ala Trp Cys               550              - #   555              - #   560                          - - CCG GTG GAG AAC GAC GTT GGC GTG CCA ACG CC - #G GCT TTC TTA AAG GCT          643                                                                       Pro Val Glu Asn Asp Val Gly Val Pro Thr Pr - #o Ala Phe Leu Lys Ala           565                 5 - #70                 5 - #75                 5 -      #80                                                                              - - GCA GAA AAC TTC ACC CTC TTG GTA AAG AAC AA - #C ATC TGG TAC CCC        AAG      691                                                                    Ala Glu Asn Phe Thr Leu Leu Val Lys Asn As - #n Ile Trp Tyr Pro Lys                          585  - #               590  - #               595              - - TTT AAC TTC AGC AAG AGG AAC ATC CTC CCC AA - #C ATC ACC ACG TCC TAC          739                                                                       Phe Asn Phe Ser Lys Arg Asn Ile Leu Pro As - #n Ile Thr Thr Ser Tyr                       600      - #           605      - #           610                  - - CTC AAA TCG TGC ATT TAC AAT GCT CAA ACG GA - #T CCC TTC TGC CCC ATA          787                                                                       Leu Lys Ser Cys Ile Tyr Asn Ala Gln Thr As - #p Pro Phe Cys Pro Ile                   615          - #       620          - #       625                      - - TTC CGT CTT GGC ACA ATC GTG GGG GAC GCG GG - #A CAT AGC TTC CAG GAG          835                                                                       Phe Arg Leu Gly Thr Ile Val Gly Asp Ala Gl - #y His Ser Phe Gln Glu               630              - #   635              - #   640                          - - ATG GCA GTT GAG GGA GGC ATC ATG GGT ATC CA - #G ATC AAG TGG GAC TGC          883                                                                       Met Ala Val Glu Gly Gly Ile Met Gly Ile Gl - #n Ile Lys Trp Asp Cys           645                 6 - #50                 6 - #55                 6 -      #60                                                                              - - AAC CTG GAT AGA GCC GCC TCC CTT TGC CTG CC - #C AGA TAT TCC TTC        CGG      931                                                                    Asn Leu Asp Arg Ala Ala Ser Leu Cys Leu Pr - #o Arg Tyr Ser Phe Arg                          665  - #               670  - #               675              - - CGC CTG GAC ACC CGG GAC CTG GAA CAC AAT GT - #G TCT CCT GGC TAC AAT          979                                                                       Arg Leu Asp Thr Arg Asp Leu Glu His Asn Va - #l Ser Pro Gly Tyr Asn                       680      - #           685      - #           690                  - - TTC AGG TTT GCC AAG TAC TAC AGG GAC CTG GC - #C GGC AAA GAG CAG CGC         1027                                                                       Phe Arg Phe Ala Lys Tyr Tyr Arg Asp Leu Al - #a Gly Lys Glu Gln Arg                   695          - #       700          - #       705                      - - ACA CTC ACC AAG GCG TAC GGC ATC CGC TTT GA - #C ATC ATC GTG TTT GGA         1075                                                                       Thr Leu Thr Lys Ala Tyr Gly Ile Arg Phe As - #p Ile Ile Val Phe Gly               710              - #   715              - #   720                          - - AAG GCT GGG AAG TTT GAC ATC ATC CCT ACC AT - #G ATC AAC GTT GGC TCT         1123                                                                       Lys Ala Gly Lys Phe Asp Ile Ile Pro Thr Me - #t Ile Asn Val Gly Ser           725                 7 - #30                 7 - #35                 7 -      #40                                                                              - - GGC TTG GCG CTC CTC GGG GTG GCG ACG GTG CT - #C TGT GAC GTC ATA        GTC     1171                                                                    Gly Leu Ala Leu Leu Gly Val Ala Thr Val Le - #u Cys Asp Val Ile Val                          745  - #               750  - #               755              - - CTC TAC TGC ATG AAG AAG AAA TAC TAC TAC CG - #G GAC AAG AAA TAT AAG         1219                                                                       Leu Tyr Cys Met Lys Lys Lys Tyr Tyr Tyr Ar - #g Asp Lys Lys Tyr Lys                       760      - #           765      - #           770                  - - TAT GTG GAA GAC TAC GAG CAG GGT CTT TCG GG - #G GAG ATG AAC CAG             1264                                                                       Tyr Val Glu Asp Tyr Glu Gln Gly Leu Ser Gl - #y Glu Met Asn Gln                       775          - #       780          - #       785                      - - TGACGCCTAA AGTTACATTT CCACCCCGCT CAGCCCGCGA AGCAGAAAGA TG -             #GGGAGAGA   1324                                                                 - - TGGCTACTGC GTCTGTCACT CTAGAGAAAG CTCCAGAGTT TCAGCTCAGT TC -            #TCCACTCC   1384                                                                 - - ACAAATACTC AGGGTTGCCA AGCACATCTT GTTGGAGCCC GGCTCTTGCT CT -            #GCTGCTCA   1444                                                                 - - GATGGGCTTC CAGATACAAG AATCCTCCTG CTTCTGCCTC TAGGAATGCT GG -            #GATCAAAC   1504                                                                 - - ATGTCACTTG CAATGCCCAT TTCCCATGGG GAGTTTGGCA TTTTTTACAT TT -            #TACCCTTT   1564                                                                 - - CCTTTTGTAT ACATCTAAGG CTGCCCTCAG ACGCAAGACG TTCTTCCACC CT -            #ATACACCC   1624                                                                 - - TTTTAATCTC ACTGTGTGTG GGAGGGGGGT CGTTTGCACA CGACGCACGG TG -            #GATGTCTG   1684                                                                 - - GTGTGCTGTT GGCTGGGCCA CCTGTGGCTT ATACAGTGTG AGCGTATGGA GG -            #TAGGAAGG   1744                                                                 - - GTCTGAGAGC AGAGACACTG CTGTGGCTTA CGGACAGGCC CAGGCTCTGT CC -            #ACGCACTT   1804                                                                 - - TATTTCTAAG GAAGGAGGCT CTCTCAGGTG CTGTCAGCAG GCCTGGGACA CC -            #ATTCCTCT   1864                                                                 - - TCCCTATAAT CAGAGAAGTT GTCCTTGTAG CAAAGGCAGG GTTAGCTTTT CC -            #TTTTATAA   1924                                                                 - - GGGCTGTGTT GAAATGACCT AGGACCAAAC ATTAAAAGAA ATAATTTTTT AA -            #AAAAAAAA   1984                                                                 - - AAAAAAAAAA AAA              - #                  - #                      - #    1997                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 7:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 388 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                           - - Met Ala Gly Cys Cys Ser Val Leu Gly Ser Ph - #e Leu Phe Glu Tyr Asp        1               5 - #                 10 - #                 15              - - Thr Pro Arg Ile Val Leu Ile Arg Ser Arg Ly - #s Val Gly Leu Met Asn                   20     - #             25     - #             30                  - - Arg Ala Val Gln Leu Leu Ile Leu Ala Tyr Va - #l Ile Gly Trp Val Phe               35         - #         40         - #         45                      - - Val Trp Glu Lys Gly Tyr Gln Glu Thr Asp Se - #r Val Val Ser Ser Val           50             - #     55             - #     60                          - - Thr Thr Lys Ala Lys Gly Val Ala Val Thr As - #n Thr Ser Gln Leu Gly       65                 - # 70                 - # 75                 - # 80       - - Phe Arg Ile Trp Asp Val Ala Asp Tyr Val Il - #e Pro Ala Gln Glu Glu                       85 - #                 90 - #                 95              - - Asn Ser Leu Phe Ile Met Thr Asn Met Ile Va - #l Thr Val Asn Gln Thr                  100      - #           105      - #           110                  - - Gln Ser Thr Cys Pro Glu Ile Pro Asp Lys Th - #r Ser Ile Cys Asn Ser              115          - #       120          - #       125                      - - Asp Ala Asp Cys Thr Pro Gly Ser Val Asp Th - #r His Ser Ser Gly Val          130              - #   135              - #   140                          - - Ala Thr Gly Arg Cys Val Pro Phe Asn Glu Se - #r Val Lys Thr Cys Glu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Val Ala Ala Trp Cys Pro Val Glu Asn Asp Va - #l Gly Val Pro Thr        Pro                                                                                             165  - #               170  - #               175             - - Ala Phe Leu Lys Ala Ala Glu Asn Phe Thr Le - #u Leu Val Lys Asn Asn                  180      - #           185      - #           190                  - - Ile Trp Tyr Pro Lys Phe Asn Phe Ser Lys Ar - #g Asn Ile Leu Pro Asn              195          - #       200          - #       205                      - - Ile Thr Thr Ser Tyr Leu Lys Ser Cys Ile Ty - #r Asn Ala Gln Thr Asp          210              - #   215              - #   220                          - - Pro Phe Cys Pro Ile Phe Arg Leu Gly Thr Il - #e Val Gly Asp Ala Gly      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - His Ser Phe Gln Glu Met Ala Val Glu Gly Gl - #y Ile Met Gly Ile        Gln                                                                                             245  - #               250  - #               255             - - Ile Lys Trp Asp Cys Asn Leu Asp Arg Ala Al - #a Ser Leu Cys Leu Pro                  260      - #           265      - #           270                  - - Arg Tyr Ser Phe Arg Arg Leu Asp Thr Arg As - #p Leu Glu His Asn Val              275          - #       280          - #       285                      - - Ser Pro Gly Tyr Asn Phe Arg Phe Ala Lys Ty - #r Tyr Arg Asp Leu Ala          290              - #   295              - #   300                          - - Gly Lys Glu Gln Arg Thr Leu Thr Lys Ala Ty - #r Gly Ile Arg Phe Asp      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ile Ile Val Phe Gly Lys Ala Gly Lys Phe As - #p Ile Ile Pro Thr        Met                                                                                             325  - #               330  - #               335             - - Ile Asn Val Gly Ser Gly Leu Ala Leu Leu Gl - #y Val Ala Thr Val Leu                  340      - #           345      - #           350                  - - Cys Asp Val Ile Val Leu Tyr Cys Met Lys Ly - #s Lys Tyr Tyr Tyr Arg              355          - #       360          - #       365                      - - Asp Lys Lys Tyr Lys Tyr Val Glu Asp Tyr Gl - #u Gln Gly Leu Ser Gly          370              - #   375              - #   380                          - - Glu Met Asn Gln                                                          385                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO: 8:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1753 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                           - - CACTGGGCTA CAGTTGCCTG GCTTACAGGA ACTGGCTCTT TTCCTCAAGC CT -             #CATTAAGC     60                                                                 - - AGCCCACTCC AGTTCTTGAT CTTTGTCTCC CAGTCCTGAA GTCCTTTCTC TC -            #CTTAGGCT    120                                                                 - - GCATCCACAG CCCTTCTAAG TGGCTGTGAG CAGTTTCTCA GT ATG AAC - # TGT ATA           174                                                                                        - #                  - #           Met Asn Cys Ile                            - #                  - #               390                   - - TCA GAC TTC TTC ACC TAC GAG ACT ACC AAG TC - #G GTG GTT GTG AAG AGC          222                                                                       Ser Asp Phe Phe Thr Tyr Glu Thr Thr Lys Se - #r Val Val Val Lys Ser                   395          - #       400          - #       405                      - - TGG ACC ATT GGG ATC ATC AAC CGA GCC GTC CA - #G CTG CTG ATT ATC TCC          270                                                                       Trp Thr Ile Gly Ile Ile Asn Arg Ala Val Gl - #n Leu Leu Ile Ile Ser               410              - #   415              - #   420                          - - TAC TTT GTG GGG TGG GTT TTC TTG CAT GAG AA - #G GCC TAC CAA GTG AGG          318                                                                       Tyr Phe Val Gly Trp Val Phe Leu His Glu Ly - #s Ala Tyr Gln Val Arg           425                 4 - #30                 4 - #35                 4 -      #40                                                                              - - GAC ACC GCC ATT GAG TCC TCA GTA GTT ACA AA - #G GTG AAA GGC TTC        GGG      366                                                                    Asp Thr Ala Ile Glu Ser Ser Val Val Thr Ly - #s Val Lys Gly Phe Gly                          445  - #               450  - #               455              - - CGC TAT GCC AAC AGA GTC ATG GAC GTG TCG GA - #T TAT GTG ACC CCA CCC          414                                                                       Arg Tyr Ala Asn Arg Val Met Asp Val Ser As - #p Tyr Val Thr Pro Pro                       460      - #           465      - #           470                  - - CAG GGC ACC TCT GTC TTT GTC ATC ATC ACC AA - #A ATG ATC GTT ACT GAA          462                                                                       Gln Gly Thr Ser Val Phe Val Ile Ile Thr Ly - #s Met Ile Val Thr Glu                   475          - #       480          - #       485                      - - AAT CAA ATG CAA GGA TTC TGT CCA GAG AAT GA - #A GAG AAG TAC CGC TGT          510                                                                       Asn Gln Met Gln Gly Phe Cys Pro Glu Asn Gl - #u Glu Lys Tyr Arg Cys               490              - #   495              - #   500                          - - GTG TCT GAC AGC CAG TGT GGG CCT GAA CGC TT - #C CCA GGT GGG GGG ATC          558                                                                       Val Ser Asp Ser Gln Cys Gly Pro Glu Arg Ph - #e Pro Gly Gly Gly Ile           505                 5 - #10                 5 - #15                 5 -      #20                                                                              - - CTC ACC GGC CGC TGC GTG AAC TAC AGC TCT GT - #T CTC CGG ACC TGT        GAG      606                                                                    Leu Thr Gly Arg Cys Val Asn Tyr Ser Ser Va - #l Leu Arg Thr Cys Glu                          525  - #               530  - #               535              - - ATC CAG GGC TGG TGC CCC ACT GAG GTG GAC AC - #C GTG GAG ATG CCT ATC          654                                                                       Ile Gln Gly Trp Cys Pro Thr Glu Val Asp Th - #r Val Glu Met Pro Ile                       540      - #           545      - #           550                  - - ATG ATG GAG GCT GAG AAC TTC ACC ATT TTC AT - #C AAG AAC AGC ATC CGT          702                                                                       Met Met Glu Ala Glu Asn Phe Thr Ile Phe Il - #e Lys Asn Ser Ile Arg                   555          - #       560          - #       565                      - - TTC CCT CTC TTC AAC TTT GAG AAG GGA AAC CT - #C CTG CCT AAC CTC ACC          750                                                                       Phe Pro Leu Phe Asn Phe Glu Lys Gly Asn Le - #u Leu Pro Asn Leu Thr               570              - #   575              - #   580                          - - GAC AAG GAC ATA AAG AGG TGC CGC TTC CAC CC - #T GAA AAG GCC CCA TTT          798                                                                       Asp Lys Asp Ile Lys Arg Cys Arg Phe His Pr - #o Glu Lys Ala Pro Phe           585                 5 - #90                 5 - #95                 6 -      #00                                                                              - - TGC CCC ATC TTG AGG GTA GGG GAT GTG GTT AA - #G TTT GCT GGA CAG        GAT      846                                                                    Cys Pro Ile Leu Arg Val Gly Asp Val Val Ly - #s Phe Ala Gly Gln Asp                          605  - #               610  - #               615              - - TTT GCC AAG CTG GCC CGC ACG GGT GGC GTT CT - #G GGT ATT AAG ATC GGC          894                                                                       Phe Ala Lys Leu Ala Arg Thr Gly Gly Val Le - #u Gly Ile Lys Ile Gly                       620      - #           625      - #           630                  - - TGG GTG TGC GAT CTA GAC AAG GCC TGG GAC CA - #G TGC ATC CCT AAA TAT          942                                                                       Trp Val Cys Asp Leu Asp Lys Ala Trp Asp Gl - #n Cys Ile Pro Lys Tyr                   635          - #       640          - #       645                      - - TCC TTC ACT CGG CTG GAT GGA GTT TCT GAG AA - #A AGC AGT GTT TCC CCT          990                                                                       Ser Phe Thr Arg Leu Asp Gly Val Ser Glu Ly - #s Ser Ser Val Ser Pro               650              - #   655              - #   660                          - - GGC TAC AAC TTC AGG TTT GCC AAA TAC TAT AA - #G ATG GAG AAC GGC AGC         1038                                                                       Gly Tyr Asn Phe Arg Phe Ala Lys Tyr Tyr Ly - #s Met Glu Asn Gly Ser           665                 6 - #70                 6 - #75                 6 -      #80                                                                              - - GAG TAC CGC ACA CTC CTG AAG GCT TTT GGC AT - #C CGC TTT GAT GTG        CTG     1086                                                                    Glu Tyr Arg Thr Leu Leu Lys Ala Phe Gly Il - #e Arg Phe Asp Val Leu                          685  - #               690  - #               695              - - GTA TAT GGG AAC GCT GGC AAG TTC AAC ATC AT - #C CCC ACC ATT ATC AGC         1134                                                                       Val Tyr Gly Asn Ala Gly Lys Phe Asn Ile Il - #e Pro Thr Ile Ile Ser                       700      - #           705      - #           710                  - - TCG GTG GCG GCC TTC ACT TCT GTG GGA GTG GG - #C ACT GTT CTC TGT GAC         1182                                                                       Ser Val Ala Ala Phe Thr Ser Val Gly Val Gl - #y Thr Val Leu Cys Asp                   715          - #       720          - #       725                      - - ATC ATC CTG CTC AAT TTC CTC AAA GGG GCT GA - #T CAC TAC AAA GCC AGG         1230                                                                       Ile Ile Leu Leu Asn Phe Leu Lys Gly Ala As - #p His Tyr Lys Ala Arg               730              - #   735              - #   740                          - - AAG TTT GAG GAG GTG ACT GAG ACA ACA CTG AA - #G GGT ACT GCG TCA ACC         1278                                                                       Lys Phe Glu Glu Val Thr Glu Thr Thr Leu Ly - #s Gly Thr Ala Ser Thr           745                 7 - #50                 7 - #55                 7 -      #60                                                                              - - AAC CCA GTG TTC GCC AGT GAC CAG GCC ACT GT - #G GAG AAG CAG TCT        ACA     1326                                                                    Asn Pro Val Phe Ala Ser Asp Gln Ala Thr Va - #l Glu Lys Gln Ser Thr                          765  - #               770  - #               775              - - GAC TCA GGG GCC TAT TCT ATT GGT CAC TAGGGCCTC - #T TCCCAGGGTT               1373                                                                       Asp Ser Gly Ala Tyr Ser Ile Gly His                                                       780      - #           785                                         - - CCATGCTCAC CCTTAGGCTG CAGAACCTGC AAACAGGCCA CTCTATCTAA GC -             #AGTCAGGG   1433                                                                 - - GTGGGAGGGG GAGAAGAAGG GCTGCTATTT CTGCTGTTCA CCCCAAAGAC TA -            #GATCCAGA   1493                                                                 - - TATCTAGGCC CTCACTGTTC AACAGATAGG CAATGCTTCC CACTAAGACT TG -            #AATCTTGC   1553                                                                 - - CTTTACCCCT TGCATGCCTC CCACCTGCTT CCCTGGATCC CAGGACAGCA GC -            #ATCCACCC   1613                                                                 - - CTTTCCAAAG GATTGAGAAA ATGGTAGCTA AGGTTACACC CATAGGACCT AC -            #CACGTACC   1673                                                                 - - AAGCACTTCC ACACATATTA TCCCTTTTCA CCCTTAAAAT AATCCTATAA GG -            #TAGAAAAA   1733                                                                 - - AAAAAAAAAA AAAAAAAAAA            - #                  - #                     175 - #3                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO: 9:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 397 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                           - - Met Asn Cys Ile Ser Asp Phe Phe Thr Tyr Gl - #u Thr Thr Lys Ser Val        1               5 - #                 10 - #                 15              - - Val Val Lys Ser Trp Thr Ile Gly Ile Ile As - #n Arg Ala Val Gln Leu                   20     - #             25     - #             30                  - - Leu Ile Ile Ser Tyr Phe Val Gly Trp Val Ph - #e Leu His Glu Lys Ala               35         - #         40         - #         45                      - - Tyr Gln Val Arg Asp Thr Ala Ile Glu Ser Se - #r Val Val Thr Lys Val           50             - #     55             - #     60                          - - Lys Gly Phe Gly Arg Tyr Ala Asn Arg Val Me - #t Asp Val Ser Asp Tyr       65                 - # 70                 - # 75                 - # 80       - - Val Thr Pro Pro Gln Gly Thr Ser Val Phe Va - #l Ile Ile Thr Lys Met                       85 - #                 90 - #                 95              - - Ile Val Thr Glu Asn Gln Met Gln Gly Phe Cy - #s Pro Glu Asn Glu Glu                  100      - #           105      - #           110                  - - Lys Tyr Arg Cys Val Ser Asp Ser Gln Cys Gl - #y Pro Glu Arg Phe Pro              115          - #       120          - #       125                      - - Gly Gly Gly Ile Leu Thr Gly Arg Cys Val As - #n Tyr Ser Ser Val Leu          130              - #   135              - #   140                          - - Arg Thr Cys Glu Ile Gln Gly Trp Cys Pro Th - #r Glu Val Asp Thr Val      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Met Pro Ile Met Met Glu Ala Glu Asn Ph - #e Thr Ile Phe Ile        Lys                                                                                             165  - #               170  - #               175             - - Asn Ser Ile Arg Phe Pro Leu Phe Asn Phe Gl - #u Lys Gly Asn Leu Leu                  180      - #           185      - #           190                  - - Pro Asn Leu Thr Asp Lys Asp Ile Lys Arg Cy - #s Arg Phe His Pro Glu              195          - #       200          - #       205                      - - Lys Ala Pro Phe Cys Pro Ile Leu Arg Val Gl - #y Asp Val Val Lys Phe          210              - #   215              - #   220                          - - Ala Gly Gln Asp Phe Ala Lys Leu Ala Arg Th - #r Gly Gly Val Leu Gly      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ile Lys Ile Gly Trp Val Cys Asp Leu Asp Ly - #s Ala Trp Asp Gln        Cys                                                                                             245  - #               250  - #               255             - - Ile Pro Lys Tyr Ser Phe Thr Arg Leu Asp Gl - #y Val Ser Glu Lys Ser                  260      - #           265      - #           270                  - - Ser Val Ser Pro Gly Tyr Asn Phe Arg Phe Al - #a Lys Tyr Tyr Lys Met              275          - #       280          - #       285                      - - Glu Asn Gly Ser Glu Tyr Arg Thr Leu Leu Ly - #s Ala Phe Gly Ile Arg          290              - #   295              - #   300                          - - Phe Asp Val Leu Val Tyr Gly Asn Ala Gly Ly - #s Phe Asn Ile Ile Pro      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Thr Ile Ile Ser Ser Val Ala Ala Phe Thr Se - #r Val Gly Val Gly        Thr                                                                                             325  - #               330  - #               335             - - Val Leu Cys Asp Ile Ile Leu Leu Asn Phe Le - #u Lys Gly Ala Asp His                  340      - #           345      - #           350                  - - Tyr Lys Ala Arg Lys Phe Glu Glu Val Thr Gl - #u Thr Thr Leu Lys Gly              355          - #       360          - #       365                      - - Thr Ala Ser Thr Asn Pro Val Phe Ala Ser As - #p Gln Ala Thr Val Glu          370              - #   375              - #   380                          - - Lys Gln Ser Thr Asp Ser Gly Ala Tyr Ser Il - #e Gly His                  385                 3 - #90                 3 - #95                            - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2643 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                          - - GCCTCCAGCT GACCTCTGGC TCCTGTCCTC TGGCTCCACC TGCACCGCCC TG -             #CTCTTCCT     60                                                                 - - AAGGGGCCAG GAAGCCCCCA GAAGCTCTAC CATCGACGTG GGTGGTGGCA CC -            #CGGCTCAC    120                                                                 - - CCTGAGAGCA GAGGGCGTGC AGGGGGCTCA GTTCTGAGCC CAGCCGGCCC AC - #C ATG           176                                                                                        - #                  - #                  - #     Met        - - GCA CGG CGG TTC CAG GAG GAG CTG GCC GCC TT - #C CTC TTC GAG TAT GAC          224                                                                       Ala Arg Arg Phe Gln Glu Glu Leu Ala Ala Ph - #e Leu Phe Glu Tyr Asp               400              - #   405              - #   410                          - - ACC CCC CGC ATG GTG CTG GTG CGT AAT AAG AA - #G GTG GGC GTT ATC TTC          272                                                                       Thr Pro Arg Met Val Leu Val Arg Asn Lys Ly - #s Val Gly Val Ile Phe           415                 4 - #20                 4 - #25                 4 -      #30                                                                              - - CGA CTG ATC CAG CTG GTG GTC CTG GTC TAC GT - #C ATC GGG TGG GTG        TTT      320                                                                    Arg Leu Ile Gln Leu Val Val Leu Val Tyr Va - #l Ile Gly Trp Val Phe                          435  - #               440  - #               445              - - CTC TAT GAG AAG GGC TAC CAG ACC TCG AGC GG - #C CTC ATC AGC AGT GTC          368                                                                       Leu Tyr Glu Lys Gly Tyr Gln Thr Ser Ser Gl - #y Leu Ile Ser Ser Val                       450      - #           455      - #           460                  - - TCT GTG AAA CTC AAG GGC CTG GCC GTG ACC CA - #G CTC CCT GGC CTC GGC          416                                                                       Ser Val Lys Leu Lys Gly Leu Ala Val Thr Gl - #n Leu Pro Gly Leu Gly                   465          - #       470          - #       475                      - - CCC CAG GTC TGG GAT GTG GCT GAC TAC GTC TT - #C CCA GCC CAG GGG GAC          464                                                                       Pro Gln Val Trp Asp Val Ala Asp Tyr Val Ph - #e Pro Ala Gln Gly Asp               480              - #   485              - #   490                          - - AAC TCC TTC GTG GTC ATG ACC AAT TTC ATC GT - #G ACC CCG AAG CAG ACT          512                                                                       Asn Ser Phe Val Val Met Thr Asn Phe Ile Va - #l Thr Pro Lys Gln Thr           495                 5 - #00                 5 - #05                 5 -      #10                                                                              - - CAA GGC TAC TGC GCA GAG CAC CCA GAA GGG GG - #C ATA TGC AAG GAA        GAC      560                                                                    Gln Gly Tyr Cys Ala Glu His Pro Glu Gly Gl - #y Ile Cys Lys Glu Asp                          515  - #               520  - #               525              - - AGT GGC TGT ACC CCT GGG AAG GCC AAG AGG AA - #G GCC CAA GGC ATC CGC          608                                                                       Ser Gly Cys Thr Pro Gly Lys Ala Lys Arg Ly - #s Ala Gln Gly Ile Arg                       530      - #           535      - #           540                  - - ACG GGC AAG TGT GTG GCC TTC AAC GAC ACT GT - #G AAG ACG TGT GAG ATC          656                                                                       Thr Gly Lys Cys Val Ala Phe Asn Asp Thr Va - #l Lys Thr Cys Glu Ile                   545          - #       550          - #       555                      - - TTT GGC TGG TGC CCC GTG GAG GTG GAT GAC GA - #C ATC CCG CGC CCT GCC          704                                                                       Phe Gly Trp Cys Pro Val Glu Val Asp Asp As - #p Ile Pro Arg Pro Ala               560              - #   565              - #   570                          - - CTT CTC CGA GAG GCC GAG AAC TTC ACT CTT TT - #C ATC AAG AAC AGC ATC          752                                                                       Leu Leu Arg Glu Ala Glu Asn Phe Thr Leu Ph - #e Ile Lys Asn Ser Ile           575                 5 - #80                 5 - #85                 5 -      #90                                                                              - - AGC TTT CCA CGC TTC AAG GTC AAC AGG CGC AA - #C CTG GTG GAG GAG        GTG      800                                                                    Ser Phe Pro Arg Phe Lys Val Asn Arg Arg As - #n Leu Val Glu Glu Val                          595  - #               600  - #               605              - - AAT GCT GCC CAC ATG AAG ACC TGC CTC TTT CA - #C AAG ACC CTG CAC CCC          848                                                                       Asn Ala Ala His Met Lys Thr Cys Leu Phe Hi - #s Lys Thr Leu His Pro                       610      - #           615      - #           620                  - - CTG TGC CCA GTC TTC CAG CTT GGC TAC GTG GT - #G CAA GAG TCA GGC CAG          896                                                                       Leu Cys Pro Val Phe Gln Leu Gly Tyr Val Va - #l Gln Glu Ser Gly Gln                   625          - #       630          - #       635                      - - AAC TTC AGC ACC CTG GCT GAG AAG GGT GGA GT - #G GTT GGC ATC ACC ATC          944                                                                       Asn Phe Ser Thr Leu Ala Glu Lys Gly Gly Va - #l Val Gly Ile Thr Ile               640              - #   645              - #   650                          - - GAC TGG CAC TGT GAC CTG GAC TGG CAC GTA CG - #G CAC TGC AGA CCC ATC          992                                                                       Asp Trp His Cys Asp Leu Asp Trp His Val Ar - #g His Cys Arg Pro Ile           655                 6 - #60                 6 - #65                 6 -      #70                                                                              - - TAT GAG TTC CAT GGG CTG TAC GAA GAG AAA AA - #T CTC TCC CCA GGC        TTC     1040                                                                    Tyr Glu Phe His Gly Leu Tyr Glu Glu Lys As - #n Leu Ser Pro Gly Phe                          675  - #               680  - #               685              - - AAC TTC AGG TTT GCC AGG CAC TTT GTG GAG AA - #C GGG ACC AAC TAC CGT         1088                                                                       Asn Phe Arg Phe Ala Arg His Phe Val Glu As - #n Gly Thr Asn Tyr Arg                       690      - #           695      - #           700                  - - CAC CTC TTC AAG GTG TTT GGG ATT CGC TTT GA - #C ATC CTG GTG GAC GGC         1136                                                                       His Leu Phe Lys Val Phe Gly Ile Arg Phe As - #p Ile Leu Val Asp Gly                   705          - #       710          - #       715                      - - AAG GCC GGG AAG TTT GAC ATC ATC CCT ACA AT - #G ACC ACC ATC GGC TCT         1184                                                                       Lys Ala Gly Lys Phe Asp Ile Ile Pro Thr Me - #t Thr Thr Ile Gly Ser               720              - #   725              - #   730                          - - GGA ATT GGC ATC TTT GGG GTG GCC ACA GTT CT - #C TGT GAC CTG CTG CTG         1232                                                                       Gly Ile Gly Ile Phe Gly Val Ala Thr Val Le - #u Cys Asp Leu Leu Leu           735                 7 - #40                 7 - #45                 7 -      #50                                                                              - - CTT CAC ATC CTG CCT AAG AGG CAC TAC TAC AA - #G CAG AAG AAG TTC        AAA     1280                                                                    Leu His Ile Leu Pro Lys Arg His Tyr Tyr Ly - #s Gln Lys Lys Phe Lys                          755  - #               760  - #               765              - - TAC GCT GAG GAC ATG GGG CCA GGG GCG GCT GA - #G CGT GAC CTC GCA GCT         1328                                                                       Tyr Ala Glu Asp Met Gly Pro Gly Ala Ala Gl - #u Arg Asp Leu Ala Ala                       770      - #           775      - #           780                  - - ACC AGC TCC ACC CTG GGC CTG CAG GAG AAC AT - #G AGG ACA TCC                 - #1370                                                                    Thr Ser Ser Thr Leu Gly Leu Gln Glu Asn Me - #t Arg Thr Ser                           785          - #       790          - #       795                      - - TGATGCTCGG GCCCCAACTC CTGACTGGGT GCAGCGTGAG GCTTCAGCCT GG -             #AGCCCTGG   1430                                                                 - - TGGGTCCCAG CCAGGGCAGA GGGGCCTCCC CAGGAAGTCT CCTACCCTCT CA -            #GCCAGGCA   1490                                                                 - - GAGAGCAGTT TGCCAGAAGC TCAGGGTGCA TAGTAGGAGA GACCTGTGCA AA -            #TCTGAGCT   1550                                                                 - - CCGGCTCCGA CCCCACACAC CCTGAGGGAG GCCTACCCTA GCCTCAGCCG CT -            #CCTGGTGG   1610                                                                 - - GGGAATGGCT GGGGGTTGGG CAGGACCCTC CCACACACCT GCACCCTAGC TT -            #CGTGCTTC   1670                                                                 - - TCTCTCCGGA CTCTCATTAT CCAACCCGCT GCCTCCATTT CTCTAGATCT GT -            #GCTCTCCG   1730                                                                 - - ATGTGGCAGT CAGTAACCAT AGGTGACTAA ATTAAACTAA AATAAAATAG AA -            #TGAAACAC   1790                                                                 - - AAAATTCAAT TCCTCGGCTG AACTAGCCAC ATTTCAACTG CTCAGTAGAT AC -            #GTGTGGTT   1850                                                                 - - AGTGGCTGCC ATACTGGACA GCTCGGGGCA TTTTCACTGT CAAAGAAAGT TC -            #TATTAGAC   1910                                                                 - - AGCCCTGCTT GAGCCCTGTT TCTTCCTGGC TTCGGTTTCC CTGGGGAACT TA -            #TCGACAAT   1970                                                                 - - GCAAGCTCCT GGGCCCACCC CCAGACCTCC TGAACCAAAA GCTCCAGGGC TG -            #GCCGTATG   2030                                                                 - - ATCTGTGTGG ATGGCAAACT CCCCAGGCCA TTCTGGGACC TAAGTTTAAG AA -            #GTGCCGTC   2090                                                                 - - CTCGAACTTT CTGACTCTAA GCTCCTGAGC GGGAGTCAGA CTTAGCCCTG AG -            #CCTGCACT   2150                                                                 - - TCCTGTTCAG GTGCAGACAC TGAACAGGGT CTCAAACACC TTCAGCATGT GT -            #GTTGTGTG   2210                                                                 - - CTCACGTGCC ACACAGTGTC TCATGCACAC AACCCAGTGT ACACACCACC TA -            #CGTGCACA   2270                                                                 - - CAGCATCCTT CCACACTGTG TATGTGAACA GCTTGGGCCC TGCAAACACA AC -            #CATCTACA   2330                                                                 - - CACATCTACA CCCCCAAGCA CACACACATG GTCCGTGCCA TGTCACCTCC AT -            #AGGGAAAG   2390                                                                 - - GCTTCTCTCC AAGTGTGCCA GGCCAGGACA GCCCTCCCAG CCATGAATCC TT -            #ACTCAGCT   2450                                                                 - - ACCTCGGGTT GGGGTGGGAG CCCCAGCCAA ATCCTGGGCT CCCTGCCTGT GG -            #CTCAGCCC   2510                                                                 - - CAGCTCCCAA GGCCTGCCTG GCTCTGTCTG AACAGAAGGT CTGGGGGAAG CG -            #AGGGGTGG   2570                                                                 - - AGTACAATAA AGGGAATGAG GACAAACAAA AAAAAAAAAA AAAAAAAAAA AA -            #AAAAAAAA   2630                                                                 - - AAAAAAAAAA AAA              - #                  - #                      - #    2643                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 399 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                          - - Met Ala Arg Arg Phe Gln Glu Glu Leu Ala Al - #a Phe Leu Phe Glu Tyr        1               5 - #                 10 - #                 15              - - Asp Thr Pro Arg Met Val Leu Val Arg Asn Ly - #s Lys Val Gly Val Ile                   20     - #             25     - #             30                  - - Phe Arg Leu Ile Gln Leu Val Val Leu Val Ty - #r Val Ile Gly Trp Val               35         - #         40         - #         45                      - - Phe Leu Tyr Glu Lys Gly Tyr Gln Thr Ser Se - #r Gly Leu Ile Ser Ser           50             - #     55             - #     60                          - - Val Ser Val Lys Leu Lys Gly Leu Ala Val Th - #r Gln Leu Pro Gly Leu       65                 - # 70                 - # 75                 - # 80       - - Gly Pro Gln Val Trp Asp Val Ala Asp Tyr Va - #l Phe Pro Ala Gln Gly                       85 - #                 90 - #                 95              - - Asp Asn Ser Phe Val Val Met Thr Asn Phe Il - #e Val Thr Pro Lys Gln                  100      - #           105      - #           110                  - - Thr Gln Gly Tyr Cys Ala Glu His Pro Glu Gl - #y Gly Ile Cys Lys Glu              115          - #       120          - #       125                      - - Asp Ser Gly Cys Thr Pro Gly Lys Ala Lys Ar - #g Lys Ala Gln Gly Ile          130              - #   135              - #   140                          - - Arg Thr Gly Lys Cys Val Ala Phe Asn Asp Th - #r Val Lys Thr Cys Glu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ile Phe Gly Trp Cys Pro Val Glu Val Asp As - #p Asp Ile Pro Arg        Pro                                                                                             165  - #               170  - #               175             - - Ala Leu Leu Arg Glu Ala Glu Asn Phe Thr Le - #u Phe Ile Lys Asn Ser                  180      - #           185      - #           190                  - - Ile Ser Phe Pro Arg Phe Lys Val Asn Arg Ar - #g Asn Leu Val Glu Glu              195          - #       200          - #       205                      - - Val Asn Ala Ala His Met Lys Thr Cys Leu Ph - #e His Lys Thr Leu His          210              - #   215              - #   220                          - - Pro Leu Cys Pro Val Phe Gln Leu Gly Tyr Va - #l Val Gln Glu Ser Gly      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Gln Asn Phe Ser Thr Leu Ala Glu Lys Gly Gl - #y Val Val Gly Ile        Thr                                                                                             245  - #               250  - #               255             - - Ile Asp Trp His Cys Asp Leu Asp Trp His Va - #l Arg His Cys Arg Pro                  260      - #           265      - #           270                  - - Ile Tyr Glu Phe His Gly Leu Tyr Glu Glu Ly - #s Asn Leu Ser Pro Gly              275          - #       280          - #       285                      - - Phe Asn Phe Arg Phe Ala Arg His Phe Val Gl - #u Asn Gly Thr Asn Tyr          290              - #   295              - #   300                          - - Arg His Leu Phe Lys Val Phe Gly Ile Arg Ph - #e Asp Ile Leu Val Asp      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Gly Lys Ala Gly Lys Phe Asp Ile Ile Pro Th - #r Met Thr Thr Ile        Gly                                                                                             325  - #               330  - #               335             - - Ser Gly Ile Gly Ile Phe Gly Val Ala Thr Va - #l Leu Cys Asp Leu Leu                  340      - #           345      - #           350                  - - Leu Leu His Ile Leu Pro Lys Arg His Tyr Ty - #r Lys Gln Lys Lys Phe              355          - #       360          - #       365                      - - Lys Tyr Ala Glu Asp Met Gly Pro Gly Ala Al - #a Glu Arg Asp Leu Ala          370              - #   375              - #   380                          - - Ala Thr Ser Ser Thr Leu Gly Leu Gln Glu As - #n Met Arg Thr Ser          385                 3 - #90                 3 - #95                          __________________________________________________________________________

We claim:
 1. A recombinant or isolated DNA molecule encoding a P_(2X)receptor wherein the receptor has the amino sequence shown in SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11.
 2. A recombinant orisolated DNA molecule encoding a P_(2X) receptor wherein the receptorhas the amino sequence shown in SEQ ID NO:5 or SEQ ID NO:11.
 3. Arecombinant or isolated DNA molecule encoding a P_(2X) receptor whereinthe receptor has the amino sequence shown in SEQ ID NO:5.
 4. The DNAmolecule as claimed in any one of claims 1 to 3, which encodes areceptor having human P_(2X) receptor activity as a ligand gated ionchannel susceptible to blockade with suramin.
 5. The DNA molecule asclaimed in any one of claims 1 to 3, which is cDNA.
 6. A vectorcomprising a DNA molecule as claimed in claim
 1. 7. A host celltransformed or transfected with the vector as claimed in claim
 6. 8. Ahost cell as claimed in claim 7 which is a stably transfected mammaliancell which expresses a P_(2X) receptor active as a ligand gated ionchannel susceptible to blockade with suramin.
 9. A method for obtaininga P_(2X) receptor active as a ligand gated ion channel susceptible toblockade with suramin comprising culturing the host cell according toclaim 8 under conditions which result in the expression of said P_(2X)receptor and purifying the expressed P_(2X) receptor.
 10. A preparationof P_(2X) receptor which is free of protein with which it is naturallyassociated wherein the receptor has the amino acid sequence shown in SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11.
 11. A recombinant orisolated DNA molecule encoding a P_(2X) receptor which has thenucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 orSEQ ID NO:10.
 12. A recombinant or isolated DNA molecule encoding aP_(2X) receptor which has the nucleotide coding sequence of SEQ ID NO:4(nucleotides 210-1406), SEQ ID NO:6 (nucleotides 101-1264), SEQ ID NO:8(nucleotides 163-1353) or SEQ ID NO:10 (nucleotides 174-1370).